WO2002016570A1 - Dms:acceptor oxidoreductase - Google Patents

Abstract

A recombinant bacterial DMS:acceptor oxidoreductase, subunit amino acid sequences andencoding nucleic acids are provided. The DMS:acceptor oxidoreductase is enantioselective with respect to prochiral organic sulfoxide substrates. The DMS:acceptor oxidoreductase is useful in methods of oxidizing prochiral organic sulfides to form corresponding sulfoxides, and more particularly, to form sulfoxide (S) enantiomers which may be used in the synthesis of chiral drugs.

Description

TITLE DMS: ACCEPTOR OXIDOREDUCTASE FIELD OF THE INNENTION THIS INNENTION relates to a recombinant bacterial DMS:acceptor oxidoreductase, amino acid sequences of constituent subunits and to their encoding nucleic acids. More particularly, this invention relates to an enantioselective DMS: acceptor oxidoreductase. This invention also relates to a method of oxidizing prochiral organic sulfides to form corresponding (S) enantiomer sulfoxides, and more particularly, to form specific sulfoxide enantiomers which may be used in the synthesis of chiral drugs. This invention also provides a method of producing a mutant DMS: acceptor oxidoreductase having a desired enzymatic activity.

BACKGROUND OF THE INNENTION

It is often desirable to produce drugs having specific enantiomeric functional groups which impart a desired activity. For example, in a case such as the β-adrenergic antagonist propanolol, the (S) enantiomeric form is a 100-fold greater antagonist that the corresponding (R) enantiomer (Bevinakatti & Baherji,

1991, J. Org. Chem. 56 5372; Crosby In: Chirality in Industry vol. 1 Eds. Collins et /., (Wileyl992). Enantiomeric sulfoxides are a particularly important chemical group used in drug synthesis. Increasingly, enzymatic processes have been utilized for generating optically pure sulfoxide enantiomers as functional groups in drugs

(Hanlon et al, 1998, Microbiology 144 2247). For this purpose, it is desirable to use "enantioselective" enzymes which display a catalytic activity towards a particular optically-active component of a racemic mixture of compounds.

Alternatively, enantioselective enzymes may catalyze formation of a specific enantiomeric product from a prochiral substrate.

Bacteria are a particularly diverse source of enantioselective enzymes. For example, the dimethyl sulfoxide reductase of Rhodobacter capsulatus catalyzes enantioselective reduction of racemic sulfoxides such as methyl p-tolyl sulfoxide by removing (S)-methyl p-tolyl sulfoxide from the racemic mixture to effectively enrich for (i?)-methyl p-tolyl sulfoxide. In contrast, the homologous enzyme from Escherichia coli displays opposite enantioselectivity by enriching for (S)-methyl p-tolyl sulfoxide. Conversely, bacterial oxidoreductases have been identified which oxidize prochiral sulfide substrates to particular enantiomeric forms of corresponding sulfoxides (Hanlon et al, 1996, supra).

A strain of Rhodobacter sulfidophilum (now known as Rhodovulum sulfidophilum strain SHI) has been isolated which displays an activity that catalyzes the oxidation of dimethyl sulfide (DMS) to dimethyl sulfoxide (DMSO; Hanlon et al, 19 '4, Microbiology 140 1953). The particular enzyme responsible for this activity was subsequently purified and partially characterized (Hanlon et al, 1996, Eur. J. Biochem. 239 391). The enzyme was characterized as a DMS:acceptor oxidoreductase having a molecular mass of 152 kDa comprising α , β and γ subunits of 94 kDa, 38 kDa and 32 kDa respectively (Hanlon et al. , 1996, supra) . Furthermore, it was proposed that this DMS : acceptor oxidoreductase had a b-type haem and molybdenum associated with the 94 kDa α subunit, suggesting that this enzyme is a member of a large family of bacterial molybdoenzymes having oxidoreductase activity (reviewed by Wooton et al, 1991, Biochim. Biophys. Acta. 1057 157, for example). OBJECT OF THE INNENTION

The present inventors have realized that the DMS: acceptor oxidoreductase of R. sulfidophilum has remained relatively uncharacterized in that the respective nucleotide and amino acid sequences of the enzyme subunits have not been elucidated. Furthermore, the activity of the DMS: acceptor oxidoreductase towards prochiral substrates, and in particular, enantioselectivity, has not been demonstrated.

It is therefore an object of the invention to provide a recombinant DMS : acceptor oxidoreductase.

SUMMARY OF THE INNENTION In a first aspect, the invention provides a recombinant

DMS: acceptor oxidoreductase subunit selected from the group consisting of: (i) a recombinant α subunit; (ii) a recombinant β subunit; (iii) a recombinant δ subunit; and (iv) a recombinant γ subunit. In a second aspect, the invention provides a recombinant

DMS: acceptor oxidoreductase comprising one or more subunits selected from the group consisting of:

(i) a recombinant α subunit; (ii) a recombinant β subunit; (iii) a recombinant δ subunit and

(iv) a recombinant γ subunit.

Preferably, the recombinant DMS: acceptor oxidoreductase comprises a recombinant subunit, a recombinant β subunit and a recombinant γ subunit. In a third aspect, the present invention provides an isolated protein selected from the group consisting of:

(i) a protein which comprises an amino acid sequence set forth in SEQ ID NO: 1; (ii) a protein which comprises an amino acid sequence set forth in SEQ ID NO:2;

(iii) a protein which comprises an amino acid sequence set forth in SEQ ID NO:3; (iv) a protein which comprises an amino acid sequence set forth in SEQ ID NO:4; (v) a protein which comprises an amino acid sequence set forth in SEQ ID NO:5; (vi) a protein which comprises an amino acid sequence set forth in SEQ ID NO:6; and (vii) a protein which comprises an amino acid sequence set forth in SEQ ID NO:7.

It will be appreciated that the amino acid sequence of SEQ ID NO:l is an N-terminal sequence of an α subunit of a DMS: acceptor oxidoreductase isolated from Rhodovulum sulfidophilum. Preferably, the α subunit has an amino acid sequence according to SEQ ID NO:4.

It will be understood that in an embodiment, the sequence of SEQ ID NO: 2 is an N-terminal amino acid sequence of a β subunit of the

DMS: acceptor oxidoreductase isolated from Rhodovulum sulfidophilum.

Preferably, the β subunit has an amino acid sequence according to SEQ ID NO: 5.

The sequence of SEQ ID NO: 3 is an N-terminal amino acid sequence of a γ subunit of the DMS .'acceptor oxidoreductase isolated from Rhodovulum sulfidophilum. Preferably, the γ subunit has an amino acid sequence according to SEQ ID NO:7.

The sequence of SEQ ID NO: 6 is an amino acid sequence of a δ subunit of the DMS: acceptor oxidoreductase of the invention.

Preferably, the DMS: acceptor oxidoreductase is enantioselective with respect to a prochiral organic sulfide substrate of formula I:

R'-S-R wherein R' is aryl, heteroaryl or alkyl; S is sulfur; R is alkyl.

More preferably, the DMS: acceptor oxidoreductase is capable of catalyzing conversion of the prochiral organic sulfide substrate to an (S) enantiomer of a corresponding organic sulfoxide of formula II:

R'-S:O-R wherein R' is aryl, heteroaryl or alkyl; S is sulfur; R is alkyl; O is oxygen; and : indicates a lone electron pair at the sulfur atom. Also included within this aspect are biologically-active polypeptide and peptide fragments, variants, derivatives and homologs of the DMS ."acceptor oxidoreductase and subunits of the invention. a fourth aspect, the invention provides an isolated nucleic acid which encodes a protein according to the first, second or third aspects.

In one embodiment, the nucleic acid encoding the DMS: acceptor oxidoreductase α subunit has a nucleotide sequence as set forth in SEQ ID NO: 8.

In one embodiment, the nucleic acid encoding the DMS .'acceptor oxidoreductase β subunit has a nucleotide sequence as set forth in SEQ ID NO: 9. In one embodiment, the nucleic acid encoding the DMS: acceptor oxidoreductase δ subunit has a nucleotide sequence as set forth in SEQ ID NO: 10. In one embodiment, the nucleic acid encoding the DMS: acceptor oxidoreductase γ subunit has a nucleotide sequence as set forth in SEQ ID NO: 11.

This aspect also includes homologs and fragments of the nucleic acids of the invention. In a fifth aspect, the invention provides a method of producing a sulfoxide of formula π, said method including the steps of:

(i) combining an isolated DMS: acceptor oxidoreductase (or a subunit thereof), or a bacterium comprising said DMS: acceptor oxidoreductase (or a subunit thereof), with a prochiral sulfide of fonnula I; and

(ii) catalyzing the formation of a corresponding sulfoxide of formula II. Preferably, the sulfoxide produced at step (ii) is an (S) enantiomer of said sulfoxide of formula H Preferably, the method further includes the step of isolating the (S) enantiomer formed at step (ii).

The bacterium at step (i) may be a bacterium which naturally comprises the DMS:acceptor oxidoreductase, such as Rhodovulum sulfidophilum, strain SHI or another bacterium engineered to express a recombinant DMS : acceptor oxidoreductase of the invention. h a sixth aspect, the invention provides a method of producing a chiral drug including the steps of:

(i) producing an (S) enantiomer of a sulfoxide according to the fifth aspect; and (ii) using the (S) enantiomer produced at step (i) in the production of said chiral drug. In a seventh aspect, the present invention provides a DMS: acceptor oxidoreductase mutant having a desired enzyme activity.

In an eighth aspect, the invention provides a method of producing a

DMS: acceptor oxidoreductase mutant, said method including the step introducing one or more mutations into a DMS: acceptor oxidoreductase, a subunit thereof or into a nucleic acid of the fourth aspect, to thereby produce a DMS: acceptor oxidoreductase mutant having a desired enzyme activity.

Suitably, the desired enzyme activity is different to that of wild- type DMS: acceptor oxidoreductase. hi particular embodiments, examples of a desired enzyme activity include enantioselective reduction of N-oxides, enrichment of chiral sulfoxides from a racemic mixture by enantioselective oxidation of the sulfoxide to a sulfone and production of (R) enantiomer sulfoxides.

In a ninth aspect, the present invention provides a DMS: acceptor oxidoreductase-deficient bacterium, preferably an SHI strain of Rhodovulum sulfidophilum.

An example of a DMS acceptor oxidoreductase-deficient bacterium is Rhodovulum sulfidophilum strain SHI -CAM deposited at AGAL on August 20 2001 under accession number NM 01/23382. hi a tenth aspect, the invention provides a plasmid which comprises an isolated nucleic acid according to the fourth aspect of the invention.

Preferably, the plasmid is useful in producing a strain of DMS: acceptor oxidoreductase-deficient Rhodovulum sulfidophilum.

Preferably, the plasmid has a -2.5 kb Sall-BamHl fragment of SEQ ID NO:8.

In a particular embodiment, the plasmid is pCMOl .

In an eleventh aspect, the invention provides an expression construct comprising one or more isolated nucleic acids accordmg to the fourth aspect operably linked to one or more regulatory sequences in an expression vector. h a twelfth aspect, the invention provides a host cell transfected or transformed with the expression vector of the eleventh aspect. Preferably, the host cell is a bacterium. In a thirteenth aspect, the invention provides a method of producing a DMS: acceptor oxidoreductase mutant bacterium including the step of deleting or mutating a nucleotide sequence of a bacterial ddH operon to thereby delete or mutate one or more DMS: acceptor oxidoreductase subunits encoded by said ddH operon.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

Table 1 : Exemplary conservative amino acid substitutions.

Table 2. Inverse PCR Primers and Probe Sequences. Table 3. Inverse PCR Product Sequencing Primers.

Table 4. ddh primers with engineered Kpήi oxXbal sites.

FIG. 1 : N-terminal amino acid sequences of α (SEQ ID NO: 1), β (SEQ ID

NO:2) and γ (SEQ ID NO:3) subunits of DMS:acceptor oxidoreductase determined by amino acid sequencing of purified subunits. The standard single- letter amino acid code is used to denote each amino acid residue. Dashes (-) indicate residues the identity of which could not be unambiguously resolved.

These were ultimately determined to be cysteine residues according to the amino acid sequence of SEQ ID NO: 4 deduced from the nucleotide sequence of SEQ ID

NO:8. FIG. 2: Deduced amino acid sequence α subunit of (A) DMS: acceptor oxidoreductase (SEQ ID NO:4); (B) β subunit of DMS -.acceptor oxidoreductase

(SEQ ID NO:5); (C) δ subunit of DMS acceptor oxidoreductase (SEQ ID NO:6); and (D) γ subunit of DMS: acceptor oxidoreductase (SEQ ID NO:7).

FIG. 3: Nucleotide sequence encoding (A) α subunit of DMS: acceptor oxidoreductase (SEQ ID NO:8); (B) β subunit of DMS acceptor oxidoreductase

(SEQ ID NO:9); (C) δ subunit of DMS -.acceptor oxidoreductase (SEQ ID NO: 10); and (D) γ subunit of DMS acceptor oxidoreductase (SEQ ID NO: 11). FIG. 4. DMS: acceptor oxidoreductase (DMS dehydrogenase) operon structure. DMS dehydrogenase subunit genes , β, γ, δ (ddhA, ddhB, ddhC and ddhD respectively) are all transcribed off the same strand. FIG. 5: Plasmid map of pCMOl used to generate DMS -.acceptor oxidoreductase deficient R. sulfidophilum. Plasmid pCMOl includes (clockwise) a 385 bp EcoRI-_5αrnHI fragment of pTKm containing the R6K origin or replication (oπ^'R6K); an 1861 bp HindΩI-BamΗI fragment from Tn5 carrying kanamycin resistance (kan); a 445 bp Hαeπ fragment from pUC19 carrying lacZ' and associated multi-cloning site; a ~2.5 kb SaR-BamHI fragment of SΕQ ID NO:8 and a 760 bp Hαeπ fragment from pSUP202 carrying an RP4 mob site (mobRP4) for plasmid mobilization. Arrows indicate the direction of transcription. FIG. 6: Chiral ΗPLC elution profile recorded at 240nm. Peak 1 with a retention time of 5.3 minutes is unreacted MPTS. Peak 2 with a retention time of 17.8 minutes is (i?)-MPTSO. Peak 3 with a retention time of 18.9 minutes is (S)-

MPTSO.

DETAILED DESCRIPTION OF THE INNENTION The present invention is predicated, at least in part, on the elucidation of the amino acid and nucleotide sequences of DMS: acceptor oxidoreductase a, β, δ, and γ subunits, and also the surprising enantioselective activity of the DMS: acceptor oxidoreductase. The invention therefore provides a means whereby DMS '.acceptor oxidoreductase can be conveniently isolated in recombinant form, and hence in large quantities if required, for use in generating sulfoxide (S) enantiomers. In particular, the present invention will greatly benefit synthesis of optically-active functional groups of drugs. Further to this, the

DMS: acceptor oxidoreductase nucleic acid of the invention will be an excellent target for mutagenesis aimed at directed evolution of desired enzyme activities using the DMS:acceptor oxidoreductase nucleic acid of the invention as a starting point. By "DMS: acceptor oxidoreductase" is meant a protein enzyme which catalyzes transfer of an electron from a compound of formula I: R'-S-R wherein R' is aryl, heteroaryl or alkyl; S is sulfur; R is alkyl. As a result of electron transfer, the compound of formula I forms a corresponding sulfoxide of formula II: R'-S:O-R wherein R' is aryl, heteroaryl or alkyl; S is sulfur; R is alkyl; O is oxygen; and : indicates a lone electron pair at the sulfur atom. Examples of compounds of formula I are dimethyl sulfide (DMS), ethyl methyl sulfide (EMS), methylthiomethy methyl sulfide (MTMS), tert-butyl methyl sulfide, methionine sulfide, methoxymethyl phenyl sulfide (MMPS), ethyl

2-pyridyl sulfide (EPS) and methyl p-tolyl sulfide (MPTS), their corresponding sulfoxides of formula II being dimethyl sulfoxide (DMSO), ethyl methyl sulfoxide (EMSO) methylthiomethy methyl sulfoxide (MTMSO), tert-butyl methyl sulfoxide, methionine sulfoxide, methoxymethyl phenyl sulfoxide (MMPSO), ethyl 2-pyridyl sulfoxide (EPSO) and methyl p-tolyl sulfoxide (MPTSO) respectively.

It will be understood by persons of skill in the art that all of the above (except DMS) are examples of prochiral sulfides of formula I. Furthermore, their corresponding sulfoxides of formula II each have (S) and (R) enantiomeric forms.

Preferably, the DMS: acceptor oxidoreductase of the invention catalyzes formation of (S) enantiomeric form of the sulfoxides of formula H

It will also be appreciated that the above definition does not exclude other activities such as reductase (electron-donating) activity towards chiral N-oxides (see Hanlon et al, 1996, supra in this regard).

In light of changing nomenclature, the terms "DMS:acceptor oxidoreductase" and "DMS dehydrogenase" are used interchangeably herein to refer to the same enzyme.

The DMS: acceptor oxidoreductase or DMS dehydrogenase is encoded by the "ddH operon" that comprises ddhA, ddhB, ddhC and ddhD genes encoding the α, β, δ and γ subunit proteins respectively. The ddhA gene was formerly referred to as dsoA.

DMS acceptor oxidoreductase and subunit proteins, homologs, variants and derivatives

For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated includes within its scope native and recombinant material.

The term "recombinant" as used herein means artificially produced through human manipulation of genetic material, such as involving techniques generally falling within the scope of "recombinant DNA technology" as is well understood in the art. By "protein"is meant an amino acid polymer comprising natural and/or non-natural amino acids as are well understood in the art.

A "peptide" is a protein having no more than fifty (50) contiguous amino acids.

A "polypeptide" is a protein having greater than fifty (50) contiguous amino acids.

The peptides of SEQ ID NOS: 1, 2 and 3 are N-terminal sequences of α, β and γ subunits respectively. The complete subunit sequences are set forth in SEQ ID NOS: 4, 5 and 7 respectively. The complete δ subunit sequence is set forth in SEQ ID NO: 6. Preferably, the DMS: acceptor oxidoreductase of the invention is a mature heterotrimer comprising , β and γ subunits.

The invention also contemplates DMS: acceptor oxidoreductase polypeptide and peptide fragments.

In one embodiment, a "fragment" includes an amino acid sequence which constitutes less than 100%, but at least 10%, preferably at least 50%, more preferably at least 80% or even more preferably at least 90% of the DMS oxidoreductase of the invention, or subunit thereof.

In a particular embodiment, a "fragment" is a small peptide, for example of at least 6, preferably at least 10 and more preferably at least 20 amino acids in length. Larger fragments comprising more than one peptide are also contemplated, and may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 18 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al. (John Wiley & Sons Inc. NY, 1997), which is incorporated herein by reference.

Alternatively, peptides can be produced by digestion of a polypeptide of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcus N8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques. The aforementioned polypeptide and peptide fragments are, preferably, biologically active.

By "biologically active"is meant having an activity at least similar to that displayed by the DMS:acceptor oxidoreductase of the invention, or that of a subunit thereof. Suitably, in a quantitative sense, the biologically active fragment displays at least 5%, more preferably at least 25% and even more preferably at least 50% of the DMS: acceptor oxidoreductase activity of the invention.

Examples of peptide fragments are the Ν-terminal sequences set forth in SEQ ID ΝOS: 1, 2 and 3.

As used herein, "variant" proteins are proteins of the invention in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions). Exemplary conservative substitutions in the polypeptide may be made according to TABLE 1. Substantial changes in function are made by selecting substitutions that are less conservative than those shown in TABLE 1. Other replacements would be non-conservative substitutions and relatively fewer of these may be tolerated. Generally, the substitutions which are likely to produce the greatest changes in a polypeptide' s properties are those in which (a) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, He, Phe or Nal); (b) a cysteine or proline is substituted for, or by, any other residue; (c) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp) or (d) a residue having a bulky side chain (e.g., Phe or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala, Ser)or no side chain (e.g., Gly). The term "variant" also includes polypeptides of the invention produced from allelic variants of the sequences exemplified in this specification.

Polypeptide variants may fall within the scope of the term "polypeptide homologs".

Polypeptide homologs share at least 60%, preferably at least 70%, more preferably at least 80% and even more preferably at least 90% sequence identity with the amino acid sequences of polypeptides of the invention as hereinbefore described.

The nearest related protein sequences currently known to the inventors are those of selenate reductase: SerA has 43.5% amino acid identity; SerB has 59.6% amino acid identity and SerC has 31.0% amino acid identity.

As generally used herein, a "homolog" shares a definable nucleotide or amino acid sequence relationship with a nucleic acid or polypeptide of the invention as the case may be.

Included within the scope of homologs are "orthologs", which are functionally-related polypeptides and their encoding nucleic acids, isolated from other organisms.

Terms used herein to describe sequence relationships between respective nucleic acids and polypeptides include "comparison window",

"sequence identity", "percentage of sequence identity" and "substantial identity". Because respective nucleic acids/polypeptides may each comprise (1) only one or more portions of a complete nucleic acid polypeptide sequence that are shared by the nucleic acids/polypeptides, and (2) one or more portions which are divergent between the nucleic acids/polypeptides, sequence comparisons are typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by hitelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al, 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference.

A detailed discussion of sequence analysis can be found in Chapter 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons hie NY, 1995-1999) and in Chapter 2 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al. supra, which are incorporated herein by reference.

The term "sequence identity" is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, "sequence identity " may be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California,

USA).

As used herein, "derivative " polypeptides are polypeptides of the invention which have been altered, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art.

"Additions" of amino acids may include fusion of the polypeptides or variants thereof with other polypeptides or proteins. A particular example of such proteins is glutathione S-transferase (GST).

Other derivatives contemplated by the invention include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the polypeptides, fragments and variants of the invention. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methylacetimidate; carbamoylation of amino groups with cyanate; pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄; reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; and trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS).

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, by way of example, to a corresponding amide. The guanidine group of arginine residues may be modified by formation of heterocyclic condensation products with reagents such as 2,3- butanedione, phenylglyoxal and glyoxal.

Sulfydryl groups may be modified by methods such as performic acid oxidation to cysteic acid; formation of mercurial derivatives using 4- chloromercuriphenylsulfonic acid, 4-chloromercuribenzoate; 2-chloromercuri-4- nitrophenol, phenylmercury chloride, and other mercurials; formation of a mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; carboxymethylation with iodoacetic acid or iodoacetamide; and carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified, for example, by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfonyl halides or by oxidation with N-bromosuccinimide.

Tyrosine residues may be modified by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

The imidazole ring of a histidine residue may be modified by N- carbethoxylation with diethylpyrocarbonate or by alkylation with iodoacetic acid derivatives.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include but are not limited to, use of 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4- amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids

Recombinant polypeptides of the invention (inclusive of fragments, variants, derivatives and homologs in general) may be prepared by any suitable procedure known to those of skill in the art.

For example, the recombinant polypeptide may be prepared by a procedure including the steps of:

(i) preparing an expression construct which comprises one or more isolated nucleic acids of the invention operably linked to one or more regulatory nucleotide sequences;

(ii) transfecting or transforming a suitable host cell with the expression construct; and (iii) expressing the recombinant polypeptide(s) in said host cell. For the purposes of host cell expression, the expression construct comprises one or more isolated nucleic acids of the invention operably linked to one or more regulatory sequences present in an expression vector.

An example of recombinant α subunit expression using the commercially-available expression vector pPROEX HT and E coli. JM109 host cells is provided in detail hereinafter.

Table 4 also provides examples of primers that may be generally useful for directional cloning of nucleic acids of the invention into complementary

XbaVKpnϊ sites of an expression vector such as pPROEX HT.

An "expression vector" may be either a self-replicating extra- chromosomal vector such as a plasmid, or a vector that integrates into a host genome. Suitably, the expression vector provides said one or more regulatory nucleotide sequences.

By "operably linked" is meant that said regulatory nucleotide sequence(s) is/are positioned relative to the recombinant nucleic acid of the invention to initiate, regulate or otherwise control transcription.

Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and silencer, enhancer or activator sequences.

Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.

In a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

The expression vector may also include a fusion partner (typically provided by the expression vector) so that the recombinant polypeptide of the invention is expressed as a fusion polypeptide with said fusion partner. The main advantage of fusion partners is that they assist identification and/or purification of said fusion polypeptide.

In order to express said fusion polypeptide, it is necessary to ligate a nucleotide sequence according to the invention into the expression vector so that the translational reading frames of the fusion partner and the nucleotide sequence of the invention coincide.

Well known examples of fusion partners include, but are not limited to, glutathione-S-transferase (GST), Fc portion of human IgG, maltose binding protein (MBP) and hexahistidine (HIS₆), which are particularly useful for isolation of the fusion polypeptide by affinity chromatography. For the purposes of fusion polypeptide purification by affinity chromatography, relevant matrices for affinity chromatography are glutathione-, amylose-, and nickel- or cobalt- conjugated resins respectively. Many such matrices are available in "kit" form, such as the QIAexpress™ system (Qiagen) useful with (HIS₆) fusion partners and the Pharmaϊcia GST purification system.

Another fusion partner well known in the art is green fluorescent protein (GFP). This fusion partner serves as a fluorescent "tag" which allows the fusion polypeptide of the invention to be identified by fluorescence microscopy or by flow cytometry. The GFP tag is useful when assessing subcellular localization of the fusion polypeptide of the invention, or for isolating cells which express the fusion polypeptide of the invention. Flow cytometric methods such as fluorescence activated cell sorting (FACS) are particularly useful in this latter application.

Preferably, the fusion partners also have protease cleavage sites, such as for Factor X_a or Thrombin, which allow the relevant protease to partially digest the fusion polypeptide of the invention and thereby liberate the recombinant polypeptide of the invention therefrom. The liberated polypeptide can then be isolated from the fusion partner by subsequent chromatographic separation.

Fusion partners according to the invention also include within their scope "epitope tags", which are usually short peptide sequences for which a specific antibody is available. Well known examples of epitope tags for which specific monoclonal antibodies are readily available include c-myc, influenza virus haemagglutinin and FLAG tags.

As hereinbefore, polypeptides of the invention may be produced by culturing a host cell transformed with said expression construct comprising a nucleic acid encoding a polypeptide, or polypeptide homolog, of the invention.

The conditions appropriate for protein expression will vary with the choice of expression vector and the host cell. This is easily ascertained by one skilled in the art through routine experimentation.

Suitable host cells for expression may be prokaryotic or eukaryotic. Preferably, the host cell is a prokaryote, more preferably a bacterium. An example of a preferred bacterium is E.coli. A example of recombinant protein expression using E.co//.JM109 bacteria is provided hereinafter.

Generally, the recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in

Sambrook, et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring

Harbor Press, 1989), incorporated herein by reference, in particular Chapters 16 and 17; Chapters 10 and 16 of CURRENT PROTOCOLS IN MOLECULAR

BIOLOGY Eds. Ausubel et al, supra, incorporated herein by reference; and Chapters 1, 5 and 6 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds.

Coligan et al, supra, which is incorporated herein by reference.

DMS:acceptor oxidoreductase nucleic acids and nucleic acid homologs

The invention provides an isolated nucleic acid that encodes a

DMS: acceptor oxidoreductase, or subunit thereof, of the invention. Preferably, the isolated nucleic acid encoding an α subunit has a nucleotide sequence set forth in SEQ ID NO: 8. Preferably, the isolated nucleic acid encoding an β subunit has a nucleotide sequence set forth in SEQ ID NO: 9.

Preferably, the isolated nucleic acid encoding an δ subunit has a nucleotide sequence set forth in SEQ ID NO: 10. Preferably, the isolated nucleic acid encoding an α subunit has a nucleotide sequence set forth in SEQ ID NO: 11.

The term "nucleic acid" as used herein designates single-or double-stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA and genomic DNA. A "polynucleotide" is a nucleic acid having eighty (80) or more contiguous nucleotides, while an "oligonucleotide" has less than eighty (80) contiguous nucleotides.

Examples of oligonucleotides are provided by SEQ ID NOS: 12- 62. A "probe" may be a single or double-stranded oligonucleotide or polynucleotide, suitably labeled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.

A "primer" is usually a single-stranded oligonucleotide, preferably having 12-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template" and being extended in a template- dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™.

The present invention also contemplates fragments of isolated nucleic acids of the invention. hi one embodiment, a "nucleic acid fragment" includes a nucleotide sequence which constitutes less than 100%, but at least 5%, preferably at least 20%, more preferably at least 50% or even more preferably at least 75% of a nucleic acid of the invention. The nucleic acid fragment maybe double-stranded or single-stranded, and in particular embodiments, be used as a probe, primer or oligonucleotide as defined above. An example of a nucleic acid fragment of the invention is a ~2.5 kb SalL-BamBI fragment of SEQ ID NO:8 used in the construction of plasmid pCMOl to be described in more detail hereinafter.

The present invention also contemplates homologs of nucleic acids of the invention. A particular homolog contemplated by the present invention is an enzyme and/or encoding nucleic acid that produces (R) sulfoxide enantiomers rather than (S) sulfoxide enantiomers. hi one embodiment, nucleic acid homologs encode polypeptide homologs of the invention, inclusive of variants, fragments and derivatives thereof.

In another embodiment, nucleic acid homologs share at least 60%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90%o sequence identity with the nucleic acids of the invention. hi yet another embodiment, nucleic acid homologs hybridize to nucleic acids of the invention under at least low stringency conditions, preferably under at least medium stringency conditions and more preferably under high stringency conditions.

"Hybridize and Hybridization" is used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA-DNA, RNA-RNA or DNA-RNA hybrid. Hybrid sequences comprising complementary nucleotide sequences occur through respective base-pairing of purine bases (guanine- and adenine-containing bases) and pyrimidine bases (cytosine- and thymine- or uracil-containing bases). Modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (thiouridine and methylcytosine) may also engage in base pairing.

"Stringency" as used herein, refers to temperature and ionic strength conditions, and presence or absence of certain organic solvents and/or detergents during hybridisation. The higher the stringency, the higher will be the required level of complementarity between hybridizing nucleotide sequences. "Stringent conditions" designates those conditions under which only nucleic acid having a high frequency of complementary bases will hybridize. Reference herein to low stringency conditions includes and encompasses:-

(i) from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridisation at 42°C, and at least about 1 M to at least about 2 M salt for washing at 42°C; and

(ii) 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M

NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C, and

(i) 2xSSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at room temperature. Medium stringency conditions include and encompass :- (i) from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridisation at 42°C, and at least about 0.5

M to at least about 0.9 M salt for washing at 42°C; and (ii) 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C and

(a) 2 x SSC, 0.1% SDS; or (b) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at 42°C.

High stringency conditions include and encompass:- (i) from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridisation at 42°C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42°C;

(ii) 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C, and (a) 0.1 x SSC, 0.1% SDS; or

(b) 0.5% BSA, ImM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at a temperature in excess of 65°C for about one hour; and (iii) 0.2 x SSC, 0.1% SDS for washing at or above 68°C for about 20 minutes, hi general, washing is carried out at T_m = 69.3 + 0.41 (G + C) % = - 12°C. However, the T_m of a duplex DNA decreases by 1°C with every increase of 1 % in the number of mismatched bases.

Notwithstanding the above, stringent conditions are well known in the art, such as described in Chapters 2.9 and 2.10 of. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Ausubel et al, supra, which are herein incorporated be reference. A skilled addressee will also recognize that various factors can be manipulated to optimize the specificity of the hybridization.

Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization.

Typically, complementary nucleotide sequences are identified by blotting techniques that include a step whereby nucleotides are immobilized on a matrix (preferably a synthetic membrane such as nitrocellulose), a hybridization step, and a detection step. Southern blotting is used to identify a complementary DNA sequence; northern blotting is used to identify a complementary RNA sequence. Dot blotting and slot blotting can be used to identify complementary DNA/DNA, DNA/RNA or RNA/RNA polynucleotide sequences. Such techniques are well known by those skilled in the art, and have been described in

CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Ausubel et al, supra, at pages 2.9.1 through 2.9.20.

According to such methods, Southern blotting involves separating DNA molecules according to size by gel electrophoresis, transferring the size- separated DNA to a synthetic membrane, and hybridizing the membrane bound

DNA to a complementary nucleotide sequence. hi dot blotting and slot blotting, DNA samples are directly applied to a synthetic membrane prior to hybridization as above.

An alternative blotting step is used when identifying complementary nucleic acids in a cDNA or genomic DNA library, such as through the process of plaque or colony hybridization. Other typical examples of this procedure are described in Chapters 8-12 of MOLECULAR CLONING A Laboratory Manual, Sambrook et al, supra which are herein incorporated by reference.

Typically, the following general procedure can be used to determine hybridization conditions. Nucleic acids are blotted transferred to a synthetic membrane, as described above. A wild type nucleotide sequence of the invention is labeled as described above, and the ability of this labeled nucleic acid to hybridize with an immobilized nucleotide sequence analyzed.

A skilled addressee will recognize that a number of factors influence hybridization. The specific activity of radioactively labeled polynucleotide sequence should typically be greater than or equal to about 10⁸ dpm/mg to provide a detectable signal. A radiolabeled nucleotide sequence of specific activity 10⁸ to 10⁹ dpm mg can detect approximately 0.5 pg of DNA. It is well known in the art that sufficient DNA must be immobilized on the membrane to permit detection. It is desirable to have excess immobilized DNA, usually 10 μg. Adding an inert polymer such as 10% (w/v) dextran sulfate (MW 500,000) or polyethylene glycol 6000 during hybridization can also increase the sensitivity of hybridization (see CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al, supra at 2.10.10). To achieve meaningful results from hybridization between a nucleic acid immobilized on a membrane and a labeled nucleic acid, a sufficient amount of the labeled nucleic acid must be hybridized to the immobilized nucleic acid following washing. Washing ensures that the labeled nucleic acid is hybridized only to the immobilized nucleic acid with a desired degree of complementarity to the labeled nucleic acid.

Methods for detecting labeled nucleic acids hybridized to an immobilized nucleic acid are well known to practitioners in the art. Such methods include autoradiography, chemiluminescent, fluorescent and colorimetric detection. hi another embodiment, nucleic acid homologs of the invention may be prepared according to the following procedure: (i) obtaining a nucleic acid extract from a suitable host; and (ii) using one or more primers, which each correspond to a distinct portion of a nucleic acid of the invention, to amplify via a nucleic acid amplification technique, one or more amplification products from said nucleic acid extract.

The primers in step (ii) may be degenerate or non-degenerate as is well understood in the art.

The primers described in Table 2 are examples of primers which may be used to amplify homologs of the invention, or at least fragments thereof. Suitable nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) as for example described in Chapter 15 of Ausubel et al. supra, which is incorporated herein by reference; strand displacement amplification (SDA) as for example described in

U.S. Patent No 5,422,252 which is incorporated herein by reference; rolling circle amplification (RCA) as for example described in Liu et al, 1996, J. Am. Chem.

Soc. 118 1587 and International Publication WO 92/01813) and Lizardi et al,

(International Publication WO 97/19193) which are incorporated herein by reference; nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et α/.,1994, Biotechniques 17 1077) which is incorporated herein by reference; ligase chain reaction (LCR) as for example described in International Application WO89/09385 which is incorporated by reference herein; and Q-β replicase amplification as for example described by

Tyagi et al, 1996, Proc. Natl. Acad. Sci. USA 93 5395) which is incorporated herein by reference. As used herein, an "amplification product" refers to a nucleic acid product generated by nucleic acid amplification techniques.

Preferably, the nucleic acid sequence amplification technique is PCR.

Mutagenesis The present invention extends to mutant DMS: acceptor oxidoreductases having an altered enzymatic activity and methods of producing said mutants.

Preferably, the mutant has an altered enzymatic activity (compared to wild-type DMS .'acceptor oxidoreductase) in the form of an activity selected by a skilled person for a particular purpose, sometimes refened to as "directed evolution".

In principle, site directed mutagenesis of an encoding nucleic acid can be employed for this purpose. Such techniques are well known in the art and include Ml 3 phage-mediated, linker- or adaptor-mediated and various PCR- mediated mutagenic techniques. General methods applicable to these approaches are provided in Chapter 9 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al, supra which is incorporated herein by reference.

However, it will be appreciated by the skilled person that site- directed mutagenesis is best performed where knowledge of the amino acid residues that contribute to enzymatic activity is available, hi many cases, this information is not available, or can only be infened by molecular modelling approximations, for example.

Therefore, according to the present invention, random mutagenesis is preferred. Random mutagenesis methods include chemical modification by hydroxylamine (Ruan et al, 1997, Gene 188 35), incorporation of dNTP analogs (Zaccolo et al, 1996, J. Mol. Biol. 255 589) and PCR-based random mutagenesis such as described in Stemmer, 1994, Proc. Natl. Acad. Sci. USA 91 10747 and Shafikhani et al, 1997, Bioiechniques 23 304, each of which references is incorporated herein. It is also noted that PCR-based random mutagenesis kits are commercially available such as the Diversify™ kit (Clontech). With regard to Shafikhani et al, 1997, supra, large libraries of nucleic acids encoding randomly mutagenized enzymes (in this case subtilisin) are produced which can be screened for activity using an appropriate enzyme assay.

Therefore, in a prefened embodiment the method of mutagenesis includes the steps of: (i) introducing one or more non-synonymous mutations into one or more nucleic acids encoding a DMS: acceptor oxidoreductase subunit; (ii) producing a mutant DMS: acceptor oxidoreductase subunit from the or each nucleic acid mutated at step (i); and (iii) selecting a DMS: acceptor oxidoreductase mutant produced at step (ii) on the basis of having a desired enzyme activity.

For example, the present invention contemplates directed evolution of activities such as enantiomer-selective dehydrogenation of N-oxides, and enantioselective oxidation of chiral sulfoxides to sulfones. The invention also contemplates mutagensis of recombinant DMS: acceptor oxidoreductase to create an enzyme that produces (R) enantiomers instead of (S) sulfoxide enantiomers.

DMS:acceptor oxidoreductase-deficient bacteria

The present invention provides bacteria which are DMS:acceptor oxidoreductase-deficient and methods for producing same.

Preferably, the bacteria are R. sulphidophilum bacteria generated by mutation, deletion or disruption of the ddH operon encoding the DMS: acceptor oxidoreductase protein subunits.

For example, the bacterial strain deposited at AGAL on August 20 2001 under accession number NM 01/23382 was created by disrupting the gene encoding the α subunit of DMS: acceptor oxidoreductase. A plasmid created for this purpose is pCMOl. Deletion of the α subunit rendered DMS: acceptor oxidoreductase-deficient R. sulphidophilum bacteria unable to utilize DMS as an electron acceptor under anaerobic conditions.

However, methods are also provided hereinafter that are applicable to disruption of any or all of the α, β, δ and γ subunit-encoding genes of the ddH operon. This method would allow production of mutant bacteria that have differentially inactive subunits as desired.

DMS: acceptor oxidoreductase-deficient bacteria of the invention may be useful in whole-cell reactions where DMS: acceptor oxidoreductase activity is unwanted. For example, such bacteria could be engineered to express another desired activity on a DMS : acceptor oxidoreductase negative background.

Antibodies

The invention also provides antibodies against the DMS: acceptor oxidoreductase polypeptides, fragments, variants, derivatives and mutants of the invention. Antibodies of the invention may be polyclonal or monoclonal.

In an embodiment, the antibody is a rabbit polyclonal antibody produced by immunization with purified α, β and γ heterotrimer as will be described in more detail hereinafter.

Well-known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al, CURRENT PROTOCOLS TN IMMUNOLOGY (John Wiley & Sons Inc. NY, 1991-1994) and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual. (Cold Spring Harbor Laboratory, 1988), which are both herein incorporated by reference.

Generally, antibodies of the invention bind to or conjugate with a polypeptide, fragment, variant or derivative of the invention. For example, the antibodies may comprise polyclonal antibodies. Such antibodies may be prepared for example by injecting a polypeptide, fragment, variant or derivative of the invention into a production species, which may include mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra, and in Harlow & Lane, 1988, supra. hi lieu of the polyclonal antisera obtained in the production species, monoclonal antibodies may be produced using the standard method as for example, described in an article by Kδhler & Milstein, 1975, Nature 256, 495, which is herein incorporated by reference, or by more recent modifications thereof as for example, described in Coligan et al, CURRENT PROTOCOLS IN

IMMUNOLOGY, supra by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the polypeptides, fragments, variants or derivatives of the invention.

The invention also includes within its scope antibodies which comprise Fc or Fab fragments of the polyclonal or monoclonal antibodies referred to above. Alternatively, the antibodies may comprise single chain Fv antibodies (scFvs) against the peptides of the invention. Such scFvs may be prepared, for example, in accordance with the methods described respectively in United States Patent No 5,091,513, European Patent No 239,400 or the article by Winter & Milstein, 1991, Nature 349 293, which are incorporated herein by reference.

The antibodies of the invention may include a label selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu³⁴), a radioisotope and a direct visual label, h the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.

A large number of enzymes suitable for use as labels is disclosed in United States Patent Specifications U.S. 4,366,241, U.S. 4,843,000, and U.S. 4,849,338, all of which are herein incorporated by reference. Suitable enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzyme label may be used alone or in combination with a second enzyme in solution.

Fluorophores may be selected from a group including fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), allophycocyanin (APC), Texas Red (TR), PerCP, Cy3, Cy5 or R-Phycoerythrin (RPE). Examples of useful fluorophores may be found, for example, in United States Patent No. 4,520,110 and United States Patent No. 4,542,104 which are herein incorporated by reference. h order that the invention may be readily understood and put into practical effect, particular prefened embodiments will now be described by way of the following non-limiting examples.

EXAMPLE 1 DMS; acceptor oxidoreductase purification DMS: acceptor oxidoreductase, was purified from whole cells of the purple non-sulfur photosynthetic bacterium Rhodovulum sulfidophilum strain

SHI as detailed below.

R. sulfidophilum strain SHI, isolated and described by Hanlon et α/.,1994, supra was grown photoautotrophically on a modified RCV (Weaver, et al, 1975, supra based medium containing 30 mM Na₂HCO₃ as a carbon source and supplemented with 20 mM DMS. Cells were incubated in completely filled 2- 1 bottles between two lights banks of two 75W tungsten light bulbs. When cell growth had reached mid-log phase the cells were harvested by centrifugation.

Subsequent to harvesting the cells were washed twice in 50 mM Tris-HCl pH 8.0, 2.5% NaCI and resuspended in 50 mM Tris-HCl pH 8.0, 1.5 mM Na_jEDTA, 0.5M sucrose to a final cell concentration of 1 g wet cells/20 ml. A periplasmic fraction was then prepared by incubation of the resuspended cells with 500 μg/ml lysozyme for 30 min at 30°C. The mixture was then centrifuged for 20 min at 9000 rpm after which the periplasmic fraction was carefully decanted. After confirming that the periplasmic fraction demonstrated DMS:DCIP dependent activity the protein was concentrated by precipitation with ammonium sulfate. DMS: acceptor oxidoreductase was precipitated when ammonium sulfate concentration was raised in two steps from 0% to 30%, the sample was then centrifuged and the pellet discarded and then from 30%> to 60%. After centrifugation the obtained pellet was resuspended in 50 mM Tris-HCl pH 8.0, 20% glycerol, the sample was dialysed three times against 10 volumes of the same buffer at 4EC. The sample was then charged onto a DEAE-Sepharose Fast Flow column (bed volume 90 ml), which had been equilibrated with 50 mM Tris-HCl pH 8.0, 20% glycerol (elution buffer). The column was washed with 5 column volumes of 170 mM NaCI that eluted the majority the periplasmic c-type cytochromes. A 500 ml linear gradient of 170-400 mM NaCI was then used to elute DMS: acceptor oxidoreductase. Peak fractions containing DMS:acceptor oxidoreductase were then pooled and charged onto a hydroxyapatite column

(BioRad) (bed volume 60 ml) equilibrated with 50 mM Tris-HCl pH 8.0, 20% glycerol. DMS:acceptor oxidoreductase was eluted with a 0-50 mM gradient of KH₂PO₄ in the same buffer. Peak fractions were pooled and concentrated by ultrafiltration (Amicon PM10 filter) and then fractionated by gel filtration on Sephacryl S200 equilibrated with 50 mM Tris-HCl, pH 8.0, 20% glycerol.

Fractions containing DMS: acceptor oxidoreductase as indicated by a deep brown colour were pooled and concentrated by ultrafiltration. EXAMPLE 2 Measurement of Purified Enzyme Activity

DMS: acceptor oxidoreductase activity was determined from the rate of electron transfer mediated by phenazine ethosulfate from DMS to dichloroindophenol (DCIP) under anaerobic conditions monitored at 600nm. This assay was performed essentially as described in Hanlon et al, 1994, supra and Hanlon et al, 1996, supra. The assay mixture contained 1 ml degassed 50 mM Tris-HCl pH 8.0 containing 1 m ol phenazine ethosulfate and 90 nmol DCIP. After addition of the enzyme to determine the rate of DMS-independent reduction of DCIP the reaction was started by addition of DMS to 20 mM from a 2 M stock solution in ethanol. DMS: acceptor oxidoreductase activity was calculated using E₆₀₀ for DCIP of 21950 M^{' 1}.

The protein concentration was determined by using the bicinchononic acid reagent with bovine serum albumin as a standard (Smith et al, 1985, Anal. Biochem. 150 76).

EXAMPLE 3 N-terminal Protein Sequencing The purified DMS: acceptor oxidoreductase was loaded onto an SDS-polyacrylamide gel which was transfened to a PVDF membrane. Individual subunits were excised from the membrane and submitted for commercial N- terminal sequencing.

Protein samples were prepared in 25% SDS, 5% β- mercaptoethanol, and 0.005% bromophenol blue. Samples were boiled at 100°C for 5 min prior to loading on a 10% SDS-polyacrylamide gel. SDS-PAGE gels were stained for protein using Coomassie Brilliant Blue R.

The α, β and γ subunits of DMS:acceptor oxidoreductase were electroblotted from a SDS-polyacrylamide gel onto a polyvinylidene fluoride membrane (BIO-RAD™). N-teraiinal amino acid sequence determination by Edman degradation was performed by the microsequencing facility at the School of Biochemistry, La Trobe University, Bundoora, Australia. The protein sequences so obtained are listed in FIG. 1 (SEQ ID NOS: 1, 2 and 3). EXAMPLE 4 DMS Dehydrogenase Operon Sequencing

On the basis of the N-terminal sequence of the alpha and beta subunits degenerate PCR primers were designed using the sequence from the start of alpha subunit to design forward primers and sequence from the start of the beta subunit to design reverse primers. These primers were the used to amplify a 2.7kb sequence of DNA encoding the alpha subunit of the DMS Dehydrogenase. This amplified product was then sequenced and the sequence conesponding to that of the mature alpha subunit was deduced, h order to sequence the genes encoding the remaimng subunits and the N-terminal signal sequence of the gene encoding the alpha subunit, inverse PCR was employed. Genomic DNA extracted from Rhodovulum sulfidophilum strain SHI was extracted and digested for investigation via Southern blotting with probes of known sequence to find fragments that could be self-ligated and sequenced. Thus the illustrated Ddh operon structure in Figure 6 was identified.

Degenerate Primer Design

N-terminal sequence obtained from the α and β subunits of the protein were then examined for regions of low degeneracy. A single forward primer from the alpha subunit sequence was designed and two reverse primers from the beta subunit were designed. The primer sequences are listed in Table 2. Degenerate PCR

These primers were employed in a PCR system based on that recommended by the Expand Long Template System (Boehringer-Mannheim). The PCR was routinely performed in a 50μL volume according to the following composition, 5μL lOx Buffer 1 (Expand Long Template System), 0.2mM each dNTP, 5% (v/v) DMSO, 125ng of each primer, 2.5U Enzyme Mix (Expand Long Template System) and 50ng template DNA and sterile distilled water to 50μL. Thermal cycling commenced with an initial five minute denaturation at 95°C followed by 30 cycles of denaturation at 94°C, 45 seconds, annealing at 50°C, ninety seconds and extension at 68°C for 4 minutes. All PCR reactions were performed in a Perkin

Elmer DNA Thermal Cycler and reactions were overlayed with 30μL mineral oil before commencement of cycling. The reaction products were analysed by agarose gel electrophoresis on a 1.0% agarose (w/v) gel for an approximate 2.7 kb reaction products. A control reaction using sterile distilled water in place of template DNA was performed. Genomic DNA Extraction

Rhodovulum sulfidophilum strain SHI was grown anaerobically overnight in RCN medium under photoautotrophic conditions. The cells were centrifuged at 8000 xg for 15 minutes in a Beckman J2-HS centrifuge and washed twice with lOmL of ice cold STE (lOOmM ΝaCl, lmM EDTA, lOmM Tris-HCl, pH 8.0) then resuspended in lOmL of STE. Lysozyme (Sigma) was added to a final concentration of 2mg.mL^"1 and the cells incubated at 37°C for three hours before the addition of lOmL STE containing 2% (w/v) SDS. Bovine pancreatic RΝase was added to a final concentration of 20μg.mL^"1 and the preparation incubated at 42°C for one hour. After the addition of proteinase K to a final concentration of 50μg.mL the preparation was incubated at 50°C until it became translucent. An equal volume of phenol/chloroform (1:1) solution was mixed with the solution and kept at room temperature for 45 minutes with gentle mixing every five minutes. The solution was centrifuged at 7000 xg for one hour at 4°C. The upper aqueous phase containing the genomic DΝA was transferred to a clean tube using a wide-bore pipette tip and mixed with 0.1 volume of 3M sodium acetate (pH 5.2) and 2.5 volumes of absolute ethanol at -20°C. The solution was mixed gently and stored at -20°C for at least one hour. The precipitated DΝA was removed from the solution using a hooked glass Pasteur pipette and washed with 70% ethanol. After air drying for five to ten minutes the DΝA was dissolved in lmL TE buffer (lOmM Tris-HCl, pH8.0, lmM EDTA) overnight at 4°C. DΝA concentration was measured spectrophotometrically by measuring the absorbance of an appropriately diluted sample between 200 and 300nm. An A₂₆₀ = 1.0 was taken to be equivalent to a DΝA concentration of SOμg.mL^"1 and a sample with an A₂₆₀/A₂₈o ratio of 1.8 was regarded as pure. Genomic DNA Restriction Enzyme Digests All restriction enzymes used were obtained from New England Biolabs or Promega. Restriction digests were routinely performed in a 20mL volume with the provided 10X Buffer for the enzyme, 5-10μg DNA, and restriction enzyme not exceeding 1U per mg of DNA and RNase to a final concentration of 2 μg.mL^"1. The reactions were incubated at 37°C for overnight.

Southern Blot

Analysis of the genomic DNA digests was perfonned by Southern blot with probes determined to bind near (within 200- nucleotides) to the restriction site of the enzyme employed. The genomic DNA digests were electrophoresed in a 0.8% agarose gel for 2 hours at 85V. The migrated genomic DNA was then transferced, by Southern blotting, to Hybond N* membrane (Pharmacia). The genomic DNA digests were then fixed to the membrane by UN cross-linking for 5 minutes. The membrane was blocked for 2 hours at 50°C in 25ml Easy-Hyb (Roche) before hybridisation for 16 hours at 50°C with 200 pmol of oligonucleotide probe. Oligonucleotide probes were 3' end-labelled with dioxygenin using the 3'

Oligonucleotide End Labeling kit (Roche). The membrane was washed before colourimetric development using ΝBT/BCIP solution (Roche). The observed banding pattern was then analysed looking for 3.0kb products or smaller suitable for use in inverse PCR reactions. The identified genomic DΝA digests producing these products were then repeated for the ligations of the genomic DΝA digest products.

Ligation of Genomic DNA

After identification of appropriate sized genomic DΝA as visualised on the Southern blot detailed above, the genomic DΝA digests were electrophoresed on a separate agarose gel. Once the appropriate sized fragments were identified, they were then gel extracted using a Qiagen Qiaquick Gel Extraction Kit. The purified genomic DΝA fragments were then routinely carried out in a lOOμL volume with lOmL of 10X Ligase buffer (Promega), 5μL of gel extracted genomic DΝA and 5U T4 DΝA Ligase (Promega). Genomic DΝA ligations were incubated overnight at 16°C. The large dilution ensures that the genomic DΝA fragments are most likely to self-ligate rather than ligating to other copies of the genomic DΝA present in solution. The ligations were ethanol precipitated the following day by adding 1 2.5 volumes of 95% ethanol and 0.1 volume 3M sodium acetate pH 4.6. The ligations were then incubated at -20°C for at least 30 minutes prior to centrifugation at 12,000 xg for 30 minutes. The supernatant was then discarded, the pellet washed with 5 volumes of ice cold 70% ethanol and the sample centrifuged again for 5 minutes at 12,000 xg. The pellet was then dried under vacuum for 10 minutes before the precipitated ligation DNA was resuspended in 30μL of sterile 50mM Tris-HCl, pH 8.0, 5mM EDTA. Inverse PCR The obtained genomic DNA samples were employed in a PCR system based on that recommended by the Expand High Fidelity System (Roche). The PCR was routinely performed in a 50mL volume according to the following composition, 5mL lOxBuffer 1 (Expand High Fidelity System), 0.2mM each dNTP, 5% (v/v) DMSO, 125ng of each primer, 2.5U Enzyme Mix (Expand High Fidelity System) and 4mL genomic ligation template DNA and sterile distilled water to 50mL.

Thermal cycling routinely commenced with an initial 5 minute denaturation at 95°C followed by 30 cycles of denaturation at 94°C, 45 seconds, annealing at 50°C, ninety seconds and extension at 68°C for 9 minutes. All PCR reactions were performed in a Perkin Elmer DNA Thermal Cycler and reactions were overlayed with 30μL mineral oil before commencement of cycling. The reaction products were analysed by agarose gel electrophoresis on a 1.0% agarose (w/v) gel for a band matching the size observed on the Southern blot. These bands were excised and gel purified using the Qiagen Qiaquick Gel Purification ldt and the obtained inverse PCR product analysed by DNA sequencing. A control reaction using sterile distilled water in place of template DNA was performed.

Design of Degenerate Sequencing Primers

Sequence data obtained from the 2.7 kb PCR product was analysed by database searches on ANGIS which identified it as being most closely related to the NarG and NarH for the forward and reverse sequences respectively. On the basis of this it was decided to continue sequencing each stand in by designing primers based on the obtained sequence data. This was repeated until the entire 2.7 kb was sequenced in both directions. The primer sequences are listed in Tables 2 and 3. Design of Inverse PCR Primers

Sequence data of the obtained genes was examined with the MAP program available on ANGIS to determine the location of the restriction sites. On the basis of these restriction sites at the 3' end of the nucleotide sequences oligonucleotides were designed for use as probes and primers. A list of the oligonucleotide sequences is presented in Tables 2 and 3. DNA Sequencing

DNA sequencing was performed using the PRISM™ Ready Reaction premix from Perkin Elmer. Unless otherwise stated reactions were performed in duplicate and both strands were sequenced. A typical reaction was performed in a 20μL volume containing lOμL of PCR product, 3.2 pmol of sequencing primer and 8μL of sequencing premix. Thermal cycling commenced with an initial 10 minute denaturation at 96°C after which the premix was added, the solution was mixed well and briefly centrifuged then covered with approximately 20μL of mineral oil. This was followed by 25 cycles of denaturation at 96°C, 30 seconds, annealing at

50°C, 15 seconds and extension at 60°C for four minutes. The reaction products were removed from under the oil and precipitated by the addition of 2.0μL 3M Sodium acetate, pH 4.6 and 50μL 95% (v/v) ethanol. This solution was well mixed and stored at -20°C for at least one hour before the reaction products were pelleted by centrifugation at maximum speed in a microcentrifuge for 20 minutes. After centrifugation the supernatant was removed and the pellet washed with 200μL of ice cold 70% (v/v) ethanol before vacuum drying. The reaction products were analysed on an Applied Biosystems 373 A automated sequencer. Assembly of DMS Dehydrogenase Subunit Sequences The various sequences for each of the putative DMS Dehydrogenase

(DMS: acceptor oxidoreductase) subunit genes were assembled on ANGIS using the Fragment Assembly program (GCG). The α, β, δ and γ subunit nucleotide sequences (SEQ ID NOS: 8-11 respectively) are shown in FIG. 3. The structure of the ddH operon is shown schematically in FIG.4. The deduced complete amino acid sequences (SEQ ID NOS: 4-7) are shown in FIG. 2. The mature heterotrimeric enzyme comprises , β and γ subunits.

The present inventors propose that the δ subunit is presumably involved in the assembly or activation of the alpha-beta complex prior to export of the alpha-beta complex to the periplasm of the bacteria. It is in the periplasm where the gamma subunit associates with the alpha-beta complex to form the mature enzyme.

EXAMPLE 5 Use of DMS dehydrogenase to produce an enantiomeric excess of a chiral sulfoxide from a prochiral sulfide The enantioselectivity of DMS dehydrogenase toward a prochiral sulfide was determined by monitoring the ratio of racemic sulfoxide products using chiral high performance liquid chromatography (HPLC). Production of Chiral Sulfoxides

The model prochiral sulfide, methyl-/?-tolyl sulfide (MPTS), was reacted with the DMS dehydrogenase employing a modified methodology of the standard enzyme activity assay. This exploited the known electron transfer pathway mediated by phenazine ethosulfate (PES) from DMS to 2,6-dichlorophenol-indophenol (DCPIP) under anaerobic conditions. The reaction mixture contained 1 ml degassed 50 mM Tris-HCl pH 8.0 containing 1 mmol PES and 90 nmol DCPIP. After addition of the enzyme the reaction was started by addition of MPTS to 2 mM, from a 2M stock solution in ethanol. Catalase was added to a final concentration of 50mg/ml to the turnover mixture to prevent any hydrogen peroxide generated during the reaction from chemically oxidising the MPTSO produced to the sulfone. The turnover mixture was incubated aerobically at room temperature for 30 hours. Analysis was performed by Chiral HPLC and Gas Chromatography Mass Spectrometry on the turnover mixture after sulfoxides were extracted into 1 volume of ethyl acetate. The oxidation of the MPTS to MPTSO was confirmed by the use of a Hewlett Packard 5890A Gas Chromatograph Mass Spectrophotometer. This detected a product of the expected mass of MPTSO. Chiral HPLC The optical purity of the sulfoxides was determined using an Alltech Chiralcel OD

HPLC column (250 X 4.6mm) eluted at a rate of 0.8ml/min with hexane/2- propanol (95:5 v/v). Detection was by UN absorption at 240nm. Methyl-/?-tolyl sulfide eluted at 5.3 min whilst the- pure enantiomers of MPTSO eluted at 17.84 min for (R)-MPTSO and 18.95 min for (S)-MPTSO (FIG. 6). The reaction products of the oxidation of MPTS were chromatographed as described for the standards. Peaks conesponding to the two racemates of MPTSO were identified from their retention time as well as their co-elution with pure MPTSO racemates in reaction mixtures that had been spiked with these standards. The calculated enantiomeric excess of 98.7% (S)-MPTSO with a yield of 22.55% MPTS converted to (S)-MPTSO.

EXAMPLE 6 Antibody production

The generation of polyclonal antibodies was performed by immunizing rabbits with 150 μg of purified DMS:acceptor oxidoreductase α, β, γ heterotrimer prepared as a 50% solution with Freund's complete adjuvant (Sigma). At subsequent two-week intervals the rabbits were boosted with an injection of a 50% solution containing Freund's incomplete adjuvant (Sigma) and

150 μg of the protein. After 3 booster injections the rabbits were test bled and the antibody trialled against the protein in a western blot. Four weeks subsequent to the test bleed the rabbits were terminally bled and approximately 60ml of blood serum obtained from each rabbit. The sera containing the polyclonal antibodies were purified by a 1 hour room temperature incubation of the blood sample after which the it was then incubated overnight at 4°C. The blood clot was removed from the sample and the solution was spun for 10 minutes at 4,000g pelleting any remaining red blood cells. The sera were then dispensed as 1ml aliquots that were placed at -70 °C for long-term storage. The final sera were tested against the DMS: acceptor oxidoreductase protein in a series of DMS: acceptor oxidoreductase polyclonal antibody titrations.

EXAMPLE 7 Recombinant Protein Expression and Purification The primers listed in Table 4 enable the cloning of nucleic acids encoding the various DMS dehydrogenase subunits either as individual subunits or as the complete operon into the commercially available pPROEX HT Prokaryotic Expression System (Invitrogen) to form an expression construct. This is performed by an XbaVKpnl double digest of the expression vector and the appropriate PCR product, amplified by the appropriate primers given in Table 4. Ligating the compatible fragment into the digested vector is then performed and the expression construct transformed into E. coli JM109 for protein expression. Aerobic growth and expression conditions were assessed in 250 ml

LB cultures supplemented with 1 mg/ml ampicillin and 1 mM NaMoO₄ in 1 litre sterile flasks. Anaerobic growth and expression analysis were carried out in sterile Schott bottles containing 600 ml of M9ZB medium consisting of 10 g/liter N-Z- amine, 5 g/liter NaCI, 1 g/liter NH₄C1, 1.5 g/liter KH₂PO₄, 15 g/liter Na^PO^ 0.25 g/liter MgSO₄.7H₂O, 0.4% glucose, supplemented with 40 mM fiimaric acid,

10-1000 mM NaMoO₄, and 30 mg/ml kanamycin. Expression under both aerobic and anaerobic conditions was induced with up to 1 mM IPTG.

An expression construct comprising the alpha subunit was transformed into E. coli JM109 for protein expression. Recombinant DMS dehydrogenase alpha subunit was purified from

E. coli JM109 cells grown aerobically at 37°C overnight in 25 ml cultures containing LB supplemented with ampicillin and used to inoculate 1 litre cultures containing M9ZB medium supplemented as described above and including NaMoO₄ at 0.5 mM and IPTG at 80 mM. The cells were grown for 24 h at room temperature and harvested by centrifugation at 5,000 xg. The cell pellets were resuspended in 500 ml of 50 mM sodium phosphate, 300 mM NaCI, pH 8.0, and frozen to lyse the cells. The frozen cells were thawed in the presence of 5 mg of DNase I and stined at room temperature until the lysate was smooth and homogeneous. The supernatant was combined with 15 - 25 ml of Ni-NTA resin, and the slurry was equilibrated with gentle stirring at 4 °C for 15 min. The slurry was poured into a 2.5 x 10-cm chromatography column, and the protein solution was allowed to flow through the resin. The column was then washed with 2 column volumes of 10 mM imidazole, 50 mM sodium phosphate, 300 mM NaCI, pH 7.5, 8 column volumes of the same solution at pH 8.0, and 3 column volumes of the pH 8 solution at a 20 mM imidazole concentration. The DMS dehydrogenase was eluted with 100 mM imidazole in 50 mM sodium phosphate, 300 mM NaCI, pH 8.0. Fractions containing the DMS dehydrogenase were combined and the protein was loaded on a 60 ml hydroxyapatite column in 50 mM Tris-HCl, pH 8.0, 20% glycerol. DMS dehydrogenase was eluted with a 0 - 50 mM gradient of KH₂PO₄ in the same buffer. Peak fractions were pooled and concentrated by ultrafiltration (Amicon PM10 filter) and then fractionated by gel filtration on Sephacryl S200 equilibrated with 50 mM Tris-HCl, pH 8.0, 20% glycerol. Fractions containing DMS oxidase were indicated by a deep brown colour and pooled for final concentration by ultrafiltration.

EXAMPLE 8 Generation of DMS: acceptor oxidoreductase alpha subunit-deficient mutant strain ofR. sulfidophilum

This example describes the production of Rhodovulum sulfidophilum strain SHI-CAM deposited at AGAL on August 20 2001 under accession number NM 01/23382.

The DMS:acceptor oxidoreductase alpha subunit gene was disrupted through a single crossover event by the insertion of the plasmid p JP5603

(pCMOl; see FIG. 5 ) into the ddhA gene on the chromosome. This was performed by first ligating a -2.5 kb SaR-BamΕl fragment of SEQ ID NO: 8 into pJP5603 and after having screened for successful insertion, the plasmid was transformed into E. coli strain SI 7-1, which allows for replication of λ pir plasmids such as pJP5603, to facilitate transfer of pCMOl into R. sulfidophilum

(as detailed below). This yielded a strain of R. sulfidophilum with a disrupted DMS: acceptor oxidoreductase alpha subunit gene.

Bacterial matings to facilitate the transfer of plasmid DNA from E. coli to R. sulfidophilum were performed essentially as previously described using the filter mating method (Masepohl et al, 1988, Mol. Gen. Genet. 212 27, which is incorporated herein by reference). Plasmid DNA to be transfened was first transformed into E. coli SI 7-1 which is capable of conjugal DNA transfer and is used as the donor strain (Simon et al, 1983, BioTechnology 1 784, which is incorporated herein by reference). R. sulfidophilum was grown anaerobically for 48 hours on TYS agar, cells from the plate were used to inoculate 10 mL of RCN medium in 250 mL conical flask. The R. sulfidophilum recipient cells were grown overnight at 30°C with vigorous shaking. The E. coli donor strain was grown overnight on LB agar containing the appropriate antibiotics. Several loopfuls of the donor strain was resuspended in 1 mL of TYS broth and gently resuspended by pipetting. 500 μL of the donor cell suspension was then mixed with an equal volume of recipient cells (R. sulfidophilum) and the cells were harvested by very gentle centrifugation at 8,000 rpm in microfuge for approximately 30 seconds.

The cell mixture was resuspended by gentle pipetting in 200 μL of TYS broth. A small disc of sterile nitrocellulose membrane (approximately 2.5 cm diameter) was placed on the TYS agar plate and the cell suspension applied to the disc. This plate was incubated aerobically at 30°C overnight before the cell mixture was resuspended in TYS broth by vortexing. The cells were plated on RCN medium containing the appropriate antibiotic to select for R. sulfidophilum cells containing the plasmid of interest and were incubated at 30°C in anaerobic jars under illumination from 75W tungsten bulbs. As R. sulfidophilum is naturally resistant to tellurite and E. coli is not (Moore & Kaplan, 1992, J. Bacteriol. 174 1505) the E. coli donor cells were killed by the addition of 20 μg.mL^"1 tellurite to the medium. Two controls were always performed; on control used the donor strain only and the other used only the recipient strain.

Plasmids with a ColΕl origin of replication cannot be maintained in R. sulfidophilum so when they are transfened into R. sulfidophilum they integrate into the chromosome. This can occur in two ways either by a single or double crossover events. In a single crossover event the plasmid integrates into the host chromosome at a single point where the plasmid and host DΝA are homologous and all the characteristics of the plasmid are retained. In this case the donor cell would be tellurite sensitive and kanamycin resistant and the recipient cells would initially be selected on the basis of tellurite resistance. A secondary screen would isolate kanamycin resistant and tellurite resistant cells that would be indicative of a single crossover event having occuned.

EXAMPLE 9 Analysis of "Si. sulfidophilum Mutant Strain The phenotype of the R. sulfidophilum mutant was confirmed by growing the mutant strain on RCN based media supplemented with 25 μg/ml kanamycin for 48 hr phototrophically. Sphaeroplasts of the cells were generated and both the periplasmic and cytoplasmic fractions were examined for the presence of the DMS: acceptor oxidoreductase using an anti-DMS: acceptor oxidoreductase antibody. Control samples used purified DMS: acceptor oxidoreductase, R. sulfidophilum periplasmic extracts, and Rhodobacter capsulatus HI 23 periplasmic extracts. The mutant strain was determined to be lacking in DMS: acceptor oxidoreductase and was also demonstrated by growth experiments to be unable to utilize DMS as an electron acceptor under anaerobic conditions. This indicated that the α subunit of DMS oxididase is essential for DMS:acceptor oxidoreductase activity as defined herein. It has also been postulated that the α subunit of DMS:acceptor oxidoreductase (or DMS:acceptor oxidoreductase as refened to in Hanlon et al, 1996, supra) alone is sufficient for a substantial portion of DMS:acceρtor oxidoreductase activity.

EXAMPLE 10 Western Blotting Detection ofR. sulfidophilum DMS.'acceptor oxidoreductase

Protein samples were electrophoresed on polyacrylamide gels. The protein was transfened to Amersham Hybond nitrocellulose membrane in a Hoefer SemiPhor semi-dry electroblotter over one hour at a cunent in mA equal to 80% of the surface area of the membrane in cm², for example, 32 mA was used for a membrane with a surface area of 40 cm². lOmM CAPS (3-[cyclohexylamino]-l- propanesulfonic acid) pH 11.0 was used as the transfer buffer.

The membrane was incubated for one hour at room temperature with gentle shaking in a blocking solution of Tris buffered saline (TBS, 20 mM Tris pH 7.5, 150mM NaCI) containing 5% (w/v) non-fat milk powder. Rabbit anti-i?. sulfidophilum DMS: acceptor oxidoreductase antibody was diluted 1:10

000 in fresh blocking solution and the membrane was incubated in this solution for one hour at room temperature with gentle shaking before washing three times in TBS for 10 minutes per wash.

Alkaline phosphatase conjugated Goat anti-rabbit IgG (Sigma) was diluted 1:2000 in TBS and incubated with the membrane for one hour at room temperature with gentle shaking. The membrane was washed three times as before except that 0.05% (v/v) Tween-20 (Sigma) was added to the TBS. The secondary antibody-alkaline phosphatase conjugate was visualised by incubation in a solution containing 100 mM Tris pH 9.5, 100 mM NaCI, 5 mM MgCl₂, 0.3 mg.mL^"1 NBT (Nitro Blue Tetrazolium), and 0.15 mg.mL^"1 BCIP (5-bromochlorindolylphosphate). The reaction was stopped by washing the membrane with water after bands became clearly visible and placing the membrane in 50 mM Tris pH 7.5, 10 mM N^EDTA.

EXAMPLE 11 Growth ø/R. sulfidophilum DMS.'acceptor oxidoreductase mutant bacteria Bacteria were grown photoautotrophically on a modified RCN (Weaver, et al. 1975, Arch. Microbiol. 105 207) based medium containing 30 mM

Νa₂HCO₃ as a carbon source and supplemented with 20 mM DMS. Cells were incubated in completely filled 2-1 bottles between two lights banks of two 75W tungsten light bulbs.

EXAMPLE 12 Generation of a Rhodovulum sulfidophilum strain with a mutated ddh Operon

DMS Dehydrogenase subunit genes can be disrupted through a double crossover event where a Spectinomycin cassette contained within a copy of the particular gene on a construct derived from the plasmid pJP5603 disrupts the ddh gene on the chromosome. The resulting strain is deficient in the ddh gene product whilst conferring the Spectinomycin resistance.

The method employed for generating a DMS Dehydrogenase (DMS: acceptor oxidoreductase) deficient strain of Rhodovulum sulfidophilum is generally applicable to any or all of the constituent genes of the ddH operon. The subunit gene is disrupted through a double crossover event where a Spectinomycin cassette contained within a copy of the gene on a construct derived from the plasmid ρJP5603 (Penfold & Pemberton, 1992, Gene 118 145) disrupts the ddh gene on the chromosome.

The constructs are generated by use of PCR primers which introduce Kpnl andXbal restricition sites at the 5' and 3' termini of the applicable ddh gene (see Table 4). The PCR amplified products are then directionally cloned into pJP5603. The ddh gene containing pJP5603 is then digested and a

Spectinomycin cassette inserted into the ddh gene. The construct is then transformed into Escherichia coli strain SI 7-1, which allows for replication of λ pir plasmids such as pJP5603, facilitating transfer of the construct into Rhodovulum sulfidophilum. This yields a strain of Rhodovulum sulfidophilum with a disrupted DMS dehydrogenase subunit gene. Bacterial matings to facilitate the transfer of plasmid DNA from E. coli to Rhodovulum sulfidophilum are performed essentially as previously described using the filter mating method (Masepohi & Klipp, 1996, Arch. Microbiol. 165 80). Plasmid DNA to be transfereed is first transformed into E. coli SI 7-1 which is capable of conjugal DNA transfer and is used as the donor strain (Simon et al, 1983, BioTechnology 1 784). Rhodovulum sulfidophilum is grown anaerobically for 48 hours on TYS agar, cells from the plate are used to inoculate lOmL of RCN medium in 250mL conical flask. The Rhodovulum sulfidophilum recipient cells are grown overnight at 30°C with vigorous shaking. The Escherichia coli donor strain is grown overnight on LB agar containing the appropriate antibiotics. Several loopfuls of the donor strain are resuspended in lmL of TYS broth and gently resuspended by pipetting. 500 μL of the donor cell suspension is then mixed with an equal volume of recipient cells (R. sulfidophilum) and the cells are harvested by very gentle centrifugation at 8,000 rpm in a microfuge for approximately 30 seconds. The cell mixture is resuspended by gentle pipetting in 200 μL of TYS broth. A small disc of sterile nitrocellulose membrane (approximately 2.5 cm diameter) is placed on the TYS agar plate and the cell suspension applied to the disc. This plate is incubated aerobically at 30°C overnight before the cell mixture is resuspended in TYS broth by vortexing. The cells are plated on RCN medium containing the appropriate antibiotic to select for R. sulfidophilum cells containing the plasmid of interest and are incubated at 30°C in anaerobic jars under illumination from 75W tungsten bulbs. As R. sulfidophilum is naturally resistant to tellurite and Escherichia coli is not (Moore & Kaplan, 1992, J. Bacteriol. 174 1505) the E. coli donor cells are killed by the addition of 20 μg.mL^"1 tellurite to the medium. Two controls are always performed; on control uses the donor strain only and the other uses only the recipient strain. Plasmids with a ColEl origin of replication cannot be maintained in R. sulfidophilum so when they are transfened into Rhodovulum sulfidophilum they integrate into the chromosome. This can occur in two ways either by a single or double crossover events. In a double crossover event the plasmid does not integrate into the host chromosome but instead the gene on the chromosome recombines with that on the plasmid where the plasmid and host DNA are homologous resulting in the ddh gene containing the Spectinomycin cassette being inserted into the Rhodovulum sulfidophilum chromosome. Consequently, the donor cells would be tellurite sensitive and kanamycin and spectinomycin resistant and the recipient cells would initially be selected on the basis of tellurite and spectinomycin resistance. A secondary screen would demonstrate kanamycin sensitivity and tellurite resistance in cells that would be indicative of a double crossover event having occuned.

Throughout the specification the aim has been to describe the prefened embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All patent and scientific literature, computer programs and algorithms refened to in this specification are incorporated herein by reference.

TABLE 1

TABLE 2

TABLE 3

TABLE 4

Claims

1. A recombinant DMS: acceptor oxidoreductase subunit selected from the group consisting of:

(i) a recombinant α subunit; (ii) a recombinant β subunit;

(iii) a recombinant δ subunit and (iv) a recombinant γ subunit.

2. A recombinant DMS:acceptor oxidoreductase comprising one or more subunits selected from the group consisting of: (i) a recombinant α subunit;

(ii) a recombinant β subunit; (iii) a recombinant δ subunit and (iv) a recombinant γ subunit.

3. A recombinant DMS: acceptor oxidoreductase heterotrimer comprising a recombinant α subunit, a recombinant β subunit and a recombinant γ subunit.

4. The recombinant DMS: acceptor oxidoreductase α subunit of Claim 1, comprising an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence set forth in SEQ ID NO: 1 ; and (ii) an amino acid sequence set forth in SEQ ID NO:4;

5. The recombinant DMS acceptor oxidoreductase β subunit of Claim 1, comprising an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence set forth in SEQ ID NO:2; and (ii) an amino acid sequence set forth in SEQ ID NO:5.

6. The recombinant DMS:acceptor oxidoreductase δ subunit of Claim 1, comprising an amino acid sequence set forth in SEQ ID NO:6.

7. The recombinant DMS:acceptor oxidoreductase γ subunit of Claim 1, comprising an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence set forth in SEQ ID NO:3; and (ii) an amino acid sequence set forth in SEQ ID NO:7.

8. The recombinant DMS'.acceptor oxidoreductase of Claim 2 or Claim 3, wherein: (A) the α subunit comprises an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence set forth in SEQ ID NO:l; and (ii) an amino acid sequence set forth in SEQ ID NO:4;

(B) the β subunit comprises an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence set forth in SEQ ID NO:2; and (ii) an amino acid sequence set forth in SEQ ID NO:5.

(C) the δ subunit comprises an amino acid sequence set forth in SEQ ID NO:6; and

(D) the γ subunit comprises an amino acid sequence selected from the group consisting of: (i) an amino acid sequence set forth in SEQ ID NO:3; and (ii) an amino acid sequence set forth in SEQ ID NO:7.

9. A biologically active fragment of the DMS:acceptor oxidoreductase α, β, δ or γ subunit of Claim 1.

10. The recombinant DMS:acceptor oxidoreductase of Claim 2 or Claim 3, which is enantioselective with respect to a prochiral organic sulfide substrate of formula I: R'-S-R wherein R' is aryl, heteroaryl or alkyl; S is sulfur; R is alkyl.

11. The recombinant DMS:acceptor oxidoreductase of Claim 10, which is capable of catalyzing conversion of the prochiral organic sulfide substrate to an

(S) enantiomer of a conesponding organic sulfoxide of formula II:

R'-S:O-R wherein R' is aryl, heteroaryl or alkyl; S is sulfur; R is alkyl; O is oxygen; : indicates a lone electron pair at the sulfur atom

12. An isolated nucleic acid that encodes the DMS:acceptor oxidoreductase subunit of Claim 1.

13. An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of:

(i) SEQ ID NO:8; (ii) SEQ ID NO:9; (iii) SEQ ID NO: 10; and

(iv) SEQ ID NO:ll.

14. An oligonucleotide derived from the isolated nucleic acid of Claim 12.

15. The oligonucleotide of Claim 14, having a nucleotide sequence according to any one of SEQ ID NOS: 12 to 62.

16. A nucleic acid homolog of an isolated nucleic acid that comprises a nucleotide sequence selected from the group consisting of:

(i) SEQ ID NO:8;

(ii) SEQ ID NO:9;

(iii) SEQ ID NO: 10; and (iv) SEQ E> NO:l l.

17. A plasmid comprising the isolated nucleic acid nucleic acid of Claim 12, or a fragment thereof.

18. The plasmid of Claim 17, wherein the fragment is a -2.5 kb Sall-BamΗI fragment of SEQ ID NO:8.

19. The plasmid of Claim 18, which is pCMOl.

20. A bacterial host cell transformed with the plasmid of Claim 17.

21. A DMS : acceptor oxidoreductase-deficient bacterial strain.

22. The bacterial strain of Claim 21, which is a DMS:acceptor oxidoreductase- deficient SHI strain of Rhodovulum sulfidophilum.

23. A DMS acceptor oxidoreductase-deficient bacterium which is

Rhodovulum sulfidophilum strain SHI -CAM deposited at AGAL on August 20 2001 under accession number NM 01/23382.

24. An expression vector comprising one or more isolated nucleic acids according to Claim 12 operably linked to one or more regulatory sequences in an expression vector.

25. A bacterial host cell transformed with the expression vector of Claim 24.

26. A method of producing a sulfoxide of formula π, said method comprising the steps of:

(i) combining an isolated DMS: acceptor oxidoreductase, or a subunit thereof, with a prochiral sulfide of formula I; and (ii) catalyzing the formation of a conesponding sulfoxide of formula H

27. The method of Claim 26, wherein the sulfoxide produced at step (ii) is an (S) enantiomer of said sulfoxide of formula H

28. The method of Claim 27, further including the step of isolating the (S) enantiomer formed at step (ii).

29. The method of Claim 26, wherein the recombinant DMS:acceptor oxidoreductase is expressed in a transformed bacterium.

30. A method of producing a chiral drug including the steps of:

(i) producing an (S) enantiomer of a sulfoxide according to Claim 27; and

(ii) using the (S) enantiomer produced at step (i) in the production of said chiral drug.

31. A DMS: acceptor oxidoreductase mutant having a desired enzyme activity different to that of wild-type DMS'.acceptor oxidoreductase.

32. The DMS:acceptor oxidoreductase mutant of Claim 31, wherein the desired enzyme activity is selected from the group consisting of: (i) enantioselective reduction of N-oxides; (ii) enrichment of chiral sulfoxides from a racemic mixture by enantioselective oxidation of the sulfoxide to a sulfone; and (iii) production of (R) enantiomer sulfoxides.

33. A method of producing a DMS:acceptor oxidoreductase mutant, said method including the step of introducing one or more non-conservative mutations into a DMS: acceptor oxidoreductase or subunit thereof, or into an isolated nucleic acid according to Claim 12, to thereby produce a DMS: acceptor oxidoreductase mutant having a desired enzyme activity different to that of a wild-type

DMS: acceptor oxidoreductase..

34. The method of Claim 33, wherein the desired enzyme activity is selected from the group consisting of:

(i) enantioselective reduction of N-oxides; (ii) enrichment of chiral sulfoxides from a racemic mixture by enantioselective oxidation of the sulfoxide to a sulfone; and

(iii) production of (R) enantiomer sulfoxides.

35. A method of producing a DMS:acceptor oxidoreductase mutant bacterium including the step of deleting or mutating a nucleotide sequence of a bacterial ddH operon to thereby delete or mutate one or more DMS: acceptor oxidoreductase subunits encoded by said ddH operon.