WO2002016570A1 - Oxydoreductase dms:accepteur - Google Patents

Oxydoreductase dms:accepteur Download PDF

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Publication number
WO2002016570A1
WO2002016570A1 PCT/AU2001/001033 AU0101033W WO0216570A1 WO 2002016570 A1 WO2002016570 A1 WO 2002016570A1 AU 0101033 W AU0101033 W AU 0101033W WO 0216570 A1 WO0216570 A1 WO 0216570A1
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dms
subunit
seq
recombinant
acceptor oxidoreductase
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PCT/AU2001/001033
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English (en)
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Christopher Adrian Mcdevitt
Alastair Graham Mcewan
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The University Of Queensland
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Priority to AU2001283688A priority Critical patent/AU2001283688A1/en
Publication of WO2002016570A1 publication Critical patent/WO2002016570A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)

Definitions

  • TITLE DMS ACCEPTOR OXIDOREDUCTASE FIELD OF THE INNENTION
  • This invention 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.
  • the (S) enantiomeric form is a 100-fold greater antagonist that the corresponding (R) enantiomer (Bevinakatti & Baherji,
  • 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
  • enantioselective enzymes may catalyze formation of a specific enantiomeric product from a prochiral substrate.
  • Bacteria are a particularly diverse source of enantioselective enzymes.
  • 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.
  • the homologous enzyme from Escherichia coli displays opposite enantioselectivity by enriching for (S)-methyl p-tolyl sulfoxide.
  • bacterial oxidoreductases have been identified which oxidize prochiral sulfide substrates to particular enantiomeric forms of corresponding sulfoxides (Hanlon et al, 1996, supra).
  • Rhodobacter sulfidophilum now known as Rhodovulum sulfidophilum strain SHI
  • Rhodovulum sulfidophilum strain SHI A strain of Rhodobacter sulfidophilum 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 ⁇ , ⁇ and ⁇ 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 ⁇ 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.
  • the invention provides a recombinant
  • DMS acceptor oxidoreductase subunit selected from the group consisting of: (i) a recombinant ⁇ subunit; (ii) a recombinant ⁇ subunit; (iii) a recombinant ⁇ subunit; and (iv) a recombinant ⁇ subunit.
  • the invention provides a recombinant
  • DMS acceptor oxidoreductase comprising one or more subunits selected from the group consisting of:
  • the recombinant DMS: acceptor oxidoreductase comprises a recombinant subunit, a recombinant ⁇ subunit and a recombinant ⁇ subunit.
  • the present invention provides an isolated protein selected from the group consisting of:
  • a protein which comprises an amino acid sequence set forth in SEQ ID NO:3 (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.
  • amino acid sequence of SEQ ID NO:l is an N-terminal sequence of an ⁇ subunit of a DMS: acceptor oxidoreductase isolated from Rhodovulum sulfidophilum.
  • the ⁇ subunit has an amino acid sequence according to SEQ ID NO:4.
  • sequence of SEQ ID NO: 2 is an N-terminal amino acid sequence of a ⁇ subunit of the
  • DMS acceptor oxidoreductase isolated from Rhodovulum sulfidophilum.
  • the ⁇ subunit has an amino acid sequence according to SEQ ID NO: 5.
  • sequence of SEQ ID NO: 3 is an N-terminal amino acid sequence of a ⁇ subunit of the DMS .'acceptor oxidoreductase isolated from Rhodovulum sulfidophilum.
  • the ⁇ subunit has an amino acid sequence according to SEQ ID NO:7.
  • sequence of SEQ ID NO: 6 is an amino acid sequence of a ⁇ subunit of the DMS: acceptor oxidoreductase of the invention.
  • 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.
  • 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.
  • biologically-active polypeptide and peptide fragments, variants, derivatives and homologs of the DMS are also included within this aspect.
  • the invention provides an isolated nucleic acid which encodes a protein according to the first, second or third aspects.
  • the nucleic acid encoding the DMS: acceptor oxidoreductase ⁇ subunit has a nucleotide sequence as set forth in SEQ ID NO: 8.
  • the nucleic acid encoding the DMS .'acceptor oxidoreductase ⁇ subunit has a nucleotide sequence as set forth in SEQ ID NO: 9. In one embodiment, the nucleic acid encoding the DMS: acceptor oxidoreductase ⁇ subunit has a nucleotide sequence as set forth in SEQ ID NO: 10. In one embodiment, the nucleic acid encoding the DMS: acceptor oxidoreductase ⁇ subunit has a nucleotide sequence as set forth in SEQ ID NO: 11.
  • the invention provides a method of producing a sulfoxide of formula ⁇ , said method including the steps of:
  • 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.
  • the invention provides a method of producing a chiral drug including the steps of:
  • the present invention provides a DMS: acceptor oxidoreductase mutant having a desired enzyme activity.
  • 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.
  • the desired enzyme activity is different to that of wild- type DMS: acceptor oxidoreductase.
  • 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.
  • the present invention provides a DMS: acceptor oxidoreductase-deficient bacterium, preferably an SHI strain of Rhodovulum sulfidophilum.
  • DMS acceptor oxidoreductase-deficient bacterium is Rhodovulum sulfidophilum strain SHI -CAM deposited at AGAL on August 20 2001 under accession number NM 01/23382.
  • the invention provides a plasmid which comprises an isolated nucleic acid according to the fourth aspect of the invention.
  • the plasmid is useful in producing a strain of DMS: acceptor oxidoreductase-deficient Rhodovulum sulfidophilum.
  • the plasmid has a -2.5 kb Sall-BamHl fragment of SEQ ID NO:8.
  • the plasmid is pCMOl .
  • 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.
  • the invention provides a host cell transfected or transformed with the expression vector of the eleventh aspect.
  • the host cell is a bacterium.
  • 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.
  • FIG. 1 N-terminal amino acid sequences of ⁇ (SEQ ID NO: 1), ⁇ (SEQ ID NO: 2), ⁇ (SEQ ID NO: 1)
  • FIG. 2 Deduced amino acid sequence ⁇ subunit of (A) DMS: acceptor oxidoreductase (SEQ ID NO:4); (B) ⁇ subunit of DMS -.acceptor oxidoreductase
  • FIG. 3 Nucleotide sequence encoding (A) ⁇ subunit of DMS: acceptor oxidoreductase (SEQ ID NO:8); (B) ⁇ subunit of DMS acceptor oxidoreductase
  • FIG. 4 DMS: acceptor oxidoreductase (DMS dehydrogenase) operon structure. DMS dehydrogenase subunit genes , ⁇ , ⁇ , ⁇ (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.
  • Plasmid pCMOl includes (clockwise) a 385 bp EcoRI-_5 ⁇ rnHI fragment of pTKm containing the R6K origin or replication (o ⁇ ' R6K); an 1861 bp Hind ⁇ I-Bam ⁇ I fragment from Tn5 carrying kanamycin resistance (kan); a 445 bp H ⁇ e ⁇ fragment from pUC19 carrying lacZ' and associated multi-cloning site; a ⁇ 2.5 kb SaR-BamHI fragment of S ⁇ Q ID NO:8 and a 760 bp H ⁇ e ⁇ fragment from pSUP202 carrying an RP4 mob site (mobRP4) for plasmid mobilization.
  • kan kanamycin resistance
  • kan kana 445 bp H ⁇ e ⁇ fragment from pUC19 carrying lacZ' and associated multi-cloning site
  • SaR-BamHI fragment of S ⁇ Q ID NO:8 and a 760 bp H ⁇ e ⁇ fragment from pSUP
  • FIG. 6 Chiral ⁇ PLC 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)-
  • the present invention is predicated, at least in part, on the elucidation of the amino acid and nucleotide sequences of DMS: acceptor oxidoreductase a, ⁇ , ⁇ , and ⁇ 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.
  • S sulfoxide
  • 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.
  • DMS acceptor oxidoreductase
  • R'-S-R wherein R' is aryl, heteroaryl or alkyl; S is sulfur; R is alkyl.
  • 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.
  • R' is aryl, heteroaryl or alkyl
  • S is sulfur
  • R is alkyl
  • O oxygen
  • : 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
  • EPS 2-pyridyl sulfide
  • MPTS methyl p-tolyl sulfide
  • DMSO dimethyl sulfoxide
  • EEO ethyl methyl sulfoxide
  • MTMSO methylthiomethy methyl sulfoxide
  • MMPSO methionine sulfoxide
  • EPSO ethyl 2-pyridyl sulfoxide
  • MPTSO methyl p-tolyl sulfoxide
  • the DMS: acceptor oxidoreductase of the invention catalyzes formation of (S) enantiomeric form of the sulfoxides of formula H
  • 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 ⁇ , ⁇ , ⁇ and ⁇ subunit proteins respectively.
  • the ddhA gene was formerly referred to as dsoA.
  • isolated 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.
  • 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.
  • 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 ⁇ , ⁇ and ⁇ subunits respectively.
  • the complete subunit sequences are set forth in SEQ ID NOS: 4, 5 and 7 respectively.
  • the complete ⁇ subunit sequence is set forth in SEQ ID NO: 6.
  • the DMS: acceptor oxidoreductase of the invention is a mature heterotrimer comprising , ⁇ and ⁇ subunits.
  • the invention also contemplates DMS: acceptor oxidoreductase polypeptide and peptide fragments.
  • 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.
  • 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.
  • 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.
  • HPLC high performance liquid chromatographic
  • 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.
  • 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.
  • peptide fragments are the ⁇ -terminal sequences set forth in SEQ ID ⁇ OS: 1, 2 and 3.
  • 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.
  • 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).
  • a hydrophilic residue e.g., Ser or Thr
  • a hydrophobic residue e.g., Ala, Leu,
  • 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.
  • SerA has 43.5% amino acid identity
  • SerB has 59.6% amino acid identity
  • SerC has 31.0% amino acid identity.
  • 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.
  • homologs are functionally-related polypeptides and their encoding nucleic acids, isolated from other organisms.
  • sequence identity means “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.
  • 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.
  • 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.
  • 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,
  • derivatives 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.
  • Additional 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).
  • 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.
  • 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 NaBH 4 ; reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; and trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS).
  • 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
  • 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 may be prepared by any suitable procedure known to those of skill in the art.
  • the recombinant polypeptide may be prepared by a procedure including the steps of:
  • 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.
  • Table 4 also provides examples of primers that may be generally useful for directional cloning of nucleic acids of the invention into complementary
  • XbaVKpn ⁇ sites of an expression vector such as pPROEX HT 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.
  • the expression vector provides said one or more regulatory nucleotide sequences.
  • 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.
  • 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.
  • promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
  • 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.
  • a fusion partner typically provided by the expression vector
  • the main advantage of fusion partners is that they assist identification and/or purification of said fusion polypeptide.
  • fusion partners include, but are not limited to, glutathione-S-transferase (GST), Fc portion of human IgG, maltose binding protein (MBP) and hexahistidine (HIS 6 ), which are particularly useful for isolation of the fusion polypeptide by affinity chromatography.
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • HIS 6 hexahistidine
  • 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 QIAexpressTM system (Qiagen) useful with (HIS 6 ) fusion partners and the Pharma ⁇ cia GST purification system.
  • GFP green fluorescent protein
  • 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.
  • 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.
  • epitope tags for which specific monoclonal antibodies are readily available include c-myc, influenza virus haemagglutinin and FLAG tags.
  • 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.
  • Suitable host cells for expression may be prokaryotic or eukaryotic.
  • 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.
  • the recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in
  • the invention provides an isolated nucleic acid that encodes a
  • DMS acceptor oxidoreductase, or subunit thereof, of the invention.
  • the isolated nucleic acid encoding an ⁇ subunit has a nucleotide sequence set forth in SEQ ID NO: 8.
  • the isolated nucleic acid encoding an ⁇ subunit has a nucleotide sequence set forth in SEQ ID NO: 9.
  • the isolated nucleic acid encoding an ⁇ subunit has a nucleotide sequence set forth in SEQ ID NO: 10.
  • the isolated nucleic acid encoding an ⁇ subunit has a nucleotide sequence set forth in SEQ ID NO: 11.
  • nucleic acid 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.
  • 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 SequenaseTM.
  • a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM.
  • 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.
  • nucleic acid homologs encode polypeptide homologs of the invention, inclusive of variants, fragments and derivatives thereof.
  • 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.
  • 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
  • modified pyrimidines thiouridine and methylcytosine
  • Stringency 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:-
  • BSA Bovine Serum Albumin
  • 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
  • BSA Bovine Serum Albumin
  • 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;
  • optimization of the stringency of the final washes can serve to ensure a high degree of hybridization.
  • 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
  • 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 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.
  • 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.
  • radioactively labeled polynucleotide sequence should typically be greater than or equal to about 10 8 dpm/mg to provide a detectable signal.
  • a radiolabeled nucleotide sequence of specific activity 10 8 to 10 9 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 ⁇ g.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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
  • nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et ⁇ /.,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- ⁇ replicase amplification as for example described by
  • an "amplification product” refers to a nucleic acid product generated by nucleic acid amplification techniques.
  • the nucleic acid sequence amplification technique is PCR.
  • Mutagenesis extends to mutant DMS: acceptor oxidoreductases having an altered enzymatic activity and methods of producing said mutants.
  • 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".
  • 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.
  • 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.
  • 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 DiversifyTM 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.
  • 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.
  • 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.
  • the present invention provides bacteria which are DMS:acceptor oxidoreductase-deficient and methods for producing same.
  • the bacteria are R. sulphidophilum bacteria generated by mutation, deletion or disruption of the ddH operon encoding the DMS: acceptor oxidoreductase protein subunits.
  • the bacterial strain deposited at AGAL on August 20 2001 under accession number NM 01/23382 was created by disrupting the gene encoding the ⁇ subunit of DMS: acceptor oxidoreductase.
  • a plasmid created for this purpose is pCMOl. Deletion of the ⁇ subunit rendered DMS: acceptor oxidoreductase-deficient R. sulphidophilum bacteria unable to utilize DMS as an electron acceptor under anaerobic conditions.
  • DMS acceptor oxidoreductase-deficient bacteria of the invention may be useful in whole-cell reactions where DMS: acceptor oxidoreductase activity is unwanted.
  • DMS acceptor oxidoreductase activity is unwanted.
  • such bacteria could be engineered to express another desired activity on a DMS : acceptor oxidoreductase negative background.
  • 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.
  • the antibody is a rabbit polyclonal antibody produced by immunization with purified ⁇ , ⁇ and ⁇ heterotrimer as will be described in more detail hereinafter.
  • antibodies of the invention bind to or conjugate with a polypeptide, fragment, variant or derivative of the invention.
  • 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.
  • monoclonal antibodies may be produced using the standard method as for example, described in an article by K ⁇ hler & 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 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.
  • the antibodies may comprise single chain Fv antibodies (scFvs) against the peptides of the invention.
  • 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 (Eu 34 ), 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 label selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu 34 ), a radioisotope and a direct visual label, h the case of a direct visual label, use may be made of a
  • Suitable enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, ⁇ -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.
  • R. sulfidophilum strain SHI isolated and described by Hanlon et ⁇ /.,1994, supra was grown photoautotrophically on a modified RCV (Weaver, et al, 1975, supra based medium containing 30 mM Na 2 HCO 3 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.
  • 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 j EDTA, 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 ⁇ g/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.
  • 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
  • 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 E 600 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).
  • Protein samples were prepared in 25% SDS, 5% ⁇ - 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.
  • 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.
  • N-terminal sequence obtained from the ⁇ and ⁇ 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.
  • PCR was routinely performed in a 50 ⁇ L volume according to the following composition, 5 ⁇ L 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 50 ⁇ L. 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
  • 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 ⁇ aCl, 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 R ⁇ ase was added to a final concentration of 20 ⁇ g.mL "1 and the preparation incubated at 42°C for one hour. After the addition of proteinase K to a final concentration of 50 ⁇ g.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 D ⁇ A 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 D ⁇ A 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 D ⁇ A was dissolved in lmL TE buffer (lOmM Tris-HCl, pH8.0, lmM EDTA) overnight at 4°C.
  • D ⁇ A concentration was measured spectrophotometrically by measuring the absorbance of an appropriately diluted sample between 200 and 300nm.
  • An A 260 1.0 was taken to be equivalent to a D ⁇ A concentration of SO ⁇ g.mL "1 and a sample with an A 260 /A 28 o 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, 5-10 ⁇ g DNA, and restriction enzyme not exceeding 1U per mg of DNA and RNase to a final concentration of 2 ⁇ g.mL "1 . The reactions were incubated at 37°C for overnight.
  • genomic DNA digests were 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'
  • genomic D ⁇ A 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 D ⁇ A fragments were then routinely carried out in a lOO ⁇ L volume with lOmL of 10X Ligase buffer (Promega), 5 ⁇ L of gel extracted genomic D ⁇ A and 5U T4 D ⁇ A Ligase (Promega). Genomic D ⁇ A ligations were incubated overnight at 16°C.
  • the large dilution ensures that the genomic D ⁇ A fragments are most likely to self-ligate rather than ligating to other copies of the genomic D ⁇ A 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 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.
  • DNA sequencing was performed using the PRISMTM 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 20 ⁇ L volume containing lO ⁇ L of PCR product, 3.2 pmol of sequencing primer and 8 ⁇ L 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 20 ⁇ L of mineral oil. This was followed by 25 cycles of denaturation at 96°C, 30 seconds, annealing at
  • reaction products were removed from under the oil and precipitated by the addition of 2.0 ⁇ L 3M Sodium acetate, pH 4.6 and 50 ⁇ L 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 200 ⁇ L 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
  • GCG Fragment Assembly program
  • the present inventors propose that the ⁇ 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.
  • PES phenazine ethosulfate
  • DCPIP 2,6-dichlorophenol-indophenol
  • 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
  • polyclonal antibodies were generated by immunizing rabbits with 150 ⁇ g of purified DMS:acceptor oxidoreductase ⁇ , ⁇ , ⁇ 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
  • the rabbits were test bled and the antibody trialled against the protein in a western blot.
  • 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
  • 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 NaMoO 4 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 KH 2 PO 4 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.
  • 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
  • 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 ⁇ L 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 ⁇ L 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.
  • E. coli is not (Moore & Kaplan, 1992, J. Bacteriol. 174 1505) the E. coli donor cells were killed by the addition of 20 ⁇ g.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 Col ⁇ l 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 D ⁇ A 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.
  • 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 ⁇ subunit of DMS oxididase is essential for DMS:acceptor oxidoreductase activity as defined herein.
  • 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 cm 2 , for example, 32 mA was used for a membrane with a surface area of 40 cm 2 .
  • 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.
  • TBS Tris buffered saline
  • Rabbit anti-i?. sulfidophilum DMS: acceptor oxidoreductase antibody was diluted 1:10
  • 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 MgCl 2 , 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.
  • 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 ⁇ JP5603 (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 is then transformed into Escherichia coli strain SI 7-1, which allows for replication of ⁇ 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.
  • 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.
  • 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 ⁇ g.mL "1 tellurite to the medium.
  • 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.
  • 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.

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Abstract

L'invention concerne une oxydoréductase DMS:accepteur bactérienne recombinée, des séquences d'acides aminés de sous-unité et les acides nucléiques codants. L'oxydoréductase DMS:accepteur est énantiosélective par rapport aux substrats sulfoxydes organiques prochiraux. L'oxydoréductase DMS:accepteur est utile dans des procédés d'oxydation de sulfures organiques prochiraux visant à former des sulfoxydes correspondants, et plus particulièrement à former des énantiomères de sulfoxyde (S) pouvant être utilisés dans la synthèse de médicaments chiraux.
PCT/AU2001/001033 2000-08-21 2001-08-21 Oxydoreductase dms:accepteur WO2002016570A1 (fr)

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AU2001283688A AU2001283688A1 (en) 2000-08-21 2001-08-21 Dms:acceptor oxidoreductase

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AUPQ9559 2000-08-21
AUPQ9559A AUPQ955900A0 (en) 2000-08-21 2000-08-21 DMS:acceptor oxidoreductase

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054123A (en) * 1995-10-27 2000-04-25 Connaught Laboratories Limited Haemophilus influenzae dimethylsulphoxide reductase enzyme

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054123A (en) * 1995-10-27 2000-04-25 Connaught Laboratories Limited Haemophilus influenzae dimethylsulphoxide reductase enzyme

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HANLON S.P. ET AL.: "Dimethylsulfide: acceptor oxidoreductase from rhodobacter sulfidophilus the purified enzyme contains beta-type heam and a pterin molybdenum cofactor", EUR. J. BIOCHEM., vol. 239, 1996, pages 391 - 396 *
HANLON S.P. ET AL.: "Isolation and characterization of a strain of rhodobacter sulfidophilus: a bacterium which grows autotrophically with dimethylsulphide as electron donor", MICROBIOLOGY, vol. 140, 1994, pages 1953 - 1958 *
HILLE R. ET AL.: "Mechanistic aspects of molybdenum-containing enzymes", FEMS MICROBIOLOGY REVIEWS, vol. 22, 1999, pages 489 - 501 *
LOOSMORE S.M. ET AL.: "Sequences of the genes encoding the A, B, and C subunits of the haemophilus influenzae dimethylsulfoxide reductase complex", GENE, vol. 169, no. 1, 22 February 1996 (1996-02-22), pages 137 - 138 *

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