WO2011130509A1 - Mutants de la l-arabinitol 4-déshydrogénase provenant de neurospora crassa - Google Patents

Mutants de la l-arabinitol 4-déshydrogénase provenant de neurospora crassa Download PDF

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WO2011130509A1
WO2011130509A1 PCT/US2011/032486 US2011032486W WO2011130509A1 WO 2011130509 A1 WO2011130509 A1 WO 2011130509A1 US 2011032486 W US2011032486 W US 2011032486W WO 2011130509 A1 WO2011130509 A1 WO 2011130509A1
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nclad
lad
nadp
nad
dehydrogenase
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Huimin Zhao
Ryan P. Sullivan
Brian Bae
Satish Nair
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The Board Of Trustees Of The University Of Illinois
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01012—L-Arabinitol 4-dehydrogenase (1.1.1.12)
    • 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.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)

Definitions

  • L-Arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12) catalyzes the second step of
  • LAD and the L-xylulose reductase that produce xylitol in the successive step are the two unique enzymes required for L-arabinose utilization, while the other enzymes in the pathway are shared in a D-xylose pathway. Due to its importance in cost-effective pentose sugar catabolism, several LADs from different fungal sources have been expressed and characterized in kinetic parameters and substrate/cofactor specificity.
  • ncLAD Neurospora crassa
  • the LAD enzyme from Neurospora crassa has previously been shown to be among the most active and stable LADs characterized.
  • ncLAD is strictly NAD + -dependent, as is the case with all LADs characterized to date, and is a member of the superfamily of medium chain dehydrogenases/reductases, with homology to xylitol dehydrogenase and sorbitol dehydrogenase.
  • the preferred substrate is L-arabinitol (compound 1 in FIG. IB), with oxidation activity also present with epimeric sugar alcohols xylitol (compound 2 in FIG. IB) and adonitol (compound 3 in FIG. IB).
  • ncLAD shows low yet detectable activity with NADP + cofactor, suggesting inherent relaxed cofactor specificity.
  • ncLAD cannot utilize D-sorbitol as a substrate, suggesting notable differences in the active site architectures between these two enzymes.
  • active site variants of ncLAD generated based on a homology model derived from the crystal structure of human sorbitol dehydrogenase, failed to demonstrate the expected kinetic parameters, further reinforcing the distinctions in the active sites of the two enzymes.
  • L-Arabinitol 4-dehydrogenase catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD + reduction. It is an essential enzyme in the development of recombinant organisms that convert L-arabinose to fuels and chemicals using the fungal L-arabinose catabolic pathway.
  • the first high-resolution X-ray crystallographic structure of an LAD from the filamentous fungi Neurospora crassa was determined to a resolution of 2.6 A.
  • the crystal structure of the ncLAD-NAD + complex was successfully utilized for the engineering of variants with altered cofactor specificity towards NADP + , These results provide the framework for engineering of variants with increased activity that can overcome the bottleneck in pentose catabolism in yeast.
  • ncLAD site-directed variants of ncLAD were created that are capable of utilizing NADP + as a cofactor, yielding the first example of a cofactor specificity switched LAD.
  • This is the first disclosure of structural data on any LAD and provides a molecular basis for understanding the existent literature on the substrate and cofactor specificity of this enzyme.
  • the engineered LAD mutants with altered cofactor specificity will be useful for applications in industrial biotechnology.
  • FIG. 1 (A) Pathway for the production of xylitol from L-arabinose in
  • FIG. 2 Ribbon diagram for the overall structure of ncLAD with bound NAD + cofactor.
  • the catalytic domain 101 houses both the catalytic and structural zinc ions; the cofactor binding domain is 102; the two zinc atoms are designated spheres 104 and the bound NAD + cofactor 103 is a ball-and-stick model.
  • FIG. 3 Xylitol production scheme using D-xylose and/or L-arabinose as starting substrate.
  • A Wild-type pathway shows inherent difficulties associated with redox cofactor imbalance.
  • B Engineered pathway is designed with an NADP + - dependent LAD to regenerate NADPH and eliminate the need for NAD + regeneration.
  • FIG. 4 Lineage and protein level mutations of the engineered LAD variants.
  • FIG. 5 NCLAD WT nucleic acid sequence (SEQ ID NO: 6) encoding amino acid sequence (SEQ ID NO: 1).
  • FIG. 6 NCLAD- S nucleic acid sequence (SEQ ID NO: 7) encoding amino acid sequence (SEQ ID NO: 2).
  • FIG. 7 NC-LAD-SPv nucleic acid sequence (SEQ ID NO: 8) encoding amino acid sequence (SEQ ID NO: 3).
  • FIG. 8 NCLAD-SRN nucleic acid sequence (SEQ ID NO: 9) encoding amino acid sequence (SEQ ID NO: 5).
  • FIG. 9 NCLAD-3X nucleic acid sequence (SEQ ID NO: 10) encoding amino acid sequence (SEQ ID NO: 4).
  • Each monomer contains a bi-domain architecture composed of a large catalytic domain (residues Ala-5 through Val-167 and Arg-308 through Leu-362) and a smaller cofactor- binding domain (residues Ala-168 through Tyr-307) with a large cleft separating the two domains (FIG. 2).
  • the smaller domain bears the structural elements of a canonical Rossman-fold and the requisite NAD + cofactor is housed within this domain.
  • the larger catalytic domain consists of an ⁇ / ⁇ fold with an overall structure similar to that found in members of the medium-chain dehydrogenase/reductase family.
  • Two bound metal ions (corresponding to zinc atoms as inferred from ICP-MS analysis) are associated with the catalytic domain, and correspond to the catalytic zinc, located at the bottom of the catalytic domain and adjacent to the NAD + cofactor, and a second zinc ion that plays a structural role. Based on inferences from other related structures, substrate binding and catalysis likely occur at the cleft between the two domains.
  • the dimer observed in the crystallographic asymmetric unit corresponds to one half of the biological tetramer and is related to the other half by the crystallographic two-fold axis. Consistent with the quaternary structures observed for other members of this structural family, the oligomeric structure may be considered a dimer of identical dimers.
  • the two monomers in the crystallographic asymmetric unit interact with the larger interface and bury 2319 A 2 of solvent-accessible surface area upon dimerization. In contrast, interaction of each monomer through the interface created by the crystallographic twofold axis buries 1908 A 2 of solvent-accessible surface area.
  • ncLAD Metal ligands - Prior characterization of ncLAD established that the purified enzyme contained 2 mol of zinc per monomer, consistent with the presence of both a structural and a catalytic metal ion.
  • the structural zinc is situated at a loop region located adjacent to the catalytic domain, where it is ligated by enzyme residues Cys-108, Cys- 111, Cys-114 and Cys-122.
  • the catalytically requisite zinc ion constitutes the second metal found in each monomer of ncLAD. This metal is coordinated by residues Cys-53, His-78, and Glu-79 with a water molecule completing a near tetrahedral coordination sphere.
  • a conserved glutamic acid residue (Glu-163) resides adjacent to the catalytic zinc and is hydrogen bonded to the zinc-bound water molecule.
  • ncLAD The promiscuity of ncLAD towards different substrates is restricted to five carbon sugars, and no activity is observed towards either D- sorbitol (compound 5 in FIG. IB) or D- mannitol. This pattern for substrate preference is similar to that observed for LADs from other sources.
  • a comparison of the active sites of the ncLAD-NAD + with that of human sorbitol dehydrogenase in complex with the inhibitor CP- 166572 provides a molecular rationale for understanding the observed substrate preference pattern.
  • a hypothetical model for the binding of the straight chain keto form of L-arabinitol to the enzyme active site can be derived from the structure of the sorbitol dehydrogenase- CP- 166572 complex. This model results in the CI and C2 carbonyl oxygen atoms directly coordinated to the zinc atom.
  • Polarization of C2 carbonyl by the zinc is likely to facilitate hydride transfer to the 2-carbon, facilitating the reduction of L-arabinitol to L- xylulose. Hydrogen-bonding between the C3 hydroxyl and Arg-308 and the C4 hydroxyl and Ser-55 further stabilizes the binding of the substrate. These hydrogen-bonding interactions are lost with either xylitol or adonitol, explaining the higher K m values observed with these other five carbon sugar substrates.
  • a number of residues flank the putative substrate-binding site above the cofactor, where steric clashes between these residues and the NAD + cofactor preclude binding of D-arabinitol, thereby confining binding to only the L- enantiomers of five carbon sugars.
  • a methionine residue protrudes inwards and caps the binding site.
  • this residue is typically a phenylalanine that is directed away from the active site (e.g. Phe-69 in sorbitol dehydrogenase).
  • loop region bearing Met-68 is extended outwards in both human sorbitol dehydrogenase and whitefly ketose reductase, resulting in a large active site cavity that can accommodate substrates such as sorbitol and the inhibitor CP- 166572.
  • adenine ring is nestled in a shallow pocket created by numerous hydrophobic residues, including Ile-212, Val-232, Thr-260, and Val-262.
  • the base of this pocket is lined by strands ⁇ 1 and ⁇ 12, and is flanked by strand ⁇ 14.
  • Both of the ribose sugars are in the 2' endo pucker, similar to that observed in the co-crystal structure of human sorbitol dehydrogenase, with the nicotinamide ring in anti conformation.
  • the nicotinamide is adjacent to the catalytic zinc ion, where it is poised for hydride transfer to the C2 atom of the substrate.
  • Asp-211 /Ile-212 are replaced by an alanine (Ala- 199) and an arginine (Arg-200), providing both a steric and electrostatic binding pocket for the - phosphate of the preferred NADP + substrate. Additional stabilization of the phosphate oxygens are provided by Arg-204 and the backbone nitrogens of Gly-178, Arg-200 and Ser-201.
  • ncLAD enzyme was subjected to site-directed and site-saturation mutagenesis of nucleic acids (SEQ ID NO: 6) encoding LAD-WT (SEQ ID NO: 1), followed by a round of error-prone PCR library screening to identify variant NADP -dependent LADs.
  • SEQ ID NO: 6 site-directed and site-saturation mutagenesis of nucleic acids
  • LAD-WT SEQ ID NO: 1
  • the kinetic parameters of the parent wild-type and engineered mutant ncLAD enzymes towards NAD + and NADP + are listed in Table 2. Three rounds of rational design were implemented to target residues Asp-211, Ile-212, and Asp-213.
  • the best first round rational design mutant D211 S (hereafter ncLAD-S (SEQ ID NO: 2; DNA disclosed as SEQ ID NO: 7)), showed a dramatic decrease in activity towards NAD + , with minimal yet detectable activity increase towards NADP + .
  • ncLAD-SRN SEQ ID NO: 5; DNA disclosed as SEQ ID NO: 9
  • Saturation mutagenesis and screening of surrounding residues Thr-210, Asp- 214, and Gly-215 did not result in finding any further improved mutants.
  • ncLAD The overall structure of ncLAD is similar to that of human sorbitol
  • R SD ketose reductase
  • Z-score 47.3; 41% sequence identity
  • RMSD L- threonine 3 -dehydrogenase
  • dehydrogenase/reductase family members including bacterial alcohol dehydrogenases and whitefly ketose reductase.
  • the orientation of the loop that harbors the structural zinc in ncLAD is nearly identical to that in human sorbitol dehydrogenase, the latter enzyme lacks the structural metal and three of the residues that bind the structural metal in ncLAD are not conserved in sorbitol dehydrogenase.
  • this loop region is involved in inter-subunit contacts that mediate oligomerization, and the presence of a stabilizing metal within this loop may play a role in the previously noted stability of ncLAD.
  • xylitol dehydrogenase from Pichia stipitis does not contain a structural zinc ion, and introduction of cysteine residues into the corresponding loop region results in an enzyme that contains an additional zinc-binding site and demonstrates improved thermostability over the wild-type.
  • catalytic zinc ion is coordinated in a tetrahedral configuration by three
  • a glutamic acid residue is analogously located in whitefly ketose reductase and human sorbitol dehydrogenase, and this residue is proposed to transiently coordinate to the zinc atom, in order to facilitate product release following catalysis. Mutation of the similar Glu-155 in rat sorbitol
  • short-chain dehydrogenases such as psXDH, are structurally and mechanistically distinct from ncLAD and other medium-chain dehydrogenases, the determinants for cofactor specificity are highly conserved.
  • ncLAD-NAD + complex crystal structure of the ncLAD-NAD + complex, provided a path for engineering ncLAD for reversed cofactor specificity.
  • a triple mutant of ncLAD (Asp-21 l ⁇ Ala/Ile- 212 ⁇ Arg/Asp-213 ⁇ Ser) was constructed based on the psXDH framework, but the resultant protein failed to express in appreciable quantities to permit biochemical analysis. Consequently, a systematic analysis of various single, double and triple mutants were carried out in order to derive an appropriate variant with switched cofactor specificity.
  • ncLAD-SR Site-directed and site-saturation mutagenesis approaches were utilized to identify a double mutant, Asp-21 l ⁇ Ser/Ile-212 ⁇ Arg (hereafter ncLAD-SR) with an expression level and solubility comparable to the wild-type enzyme.
  • ncLAD-SR double mutant was subject to an error-prone PCR library screen.
  • This approach identified a third mutation (Ser-348 ⁇ Thr) that improved upon the solubility and activity of the ncLAD-SR template.
  • This triple mutant Asp-21 l ⁇ Ser/Ile- 212 ⁇ Arg/Ser-348 ⁇ Thr shows a clear switch in co-factor preference, with a catalytic efficiency that is nearly 20-fold higher with NADP + than with NAD + .
  • Xylose reductase (XR) and L-xylulose reductase (LXR) are preferably NADPH-dependent, whereas L-arabinitol 4-dehydrogenase (LAD) is NAD + - dependent.
  • L-arabinitol 4-dehydrogenase (LAD) is NAD + - dependent.
  • NAD(H) and NADP(H) differ only in the phosphate group esterified at the - position of adenosine ribose, and therefore there are a limited number of amino acid residues interacting with this characteristic moiety that are suitable as the first candidates for protein engineering using site-directed mutagenesis.
  • the ability of polyol dehydrogenases to discriminate between NAD + and NADP + has been established to lie in the amino acid sequence of the ⁇ - ⁇ - ⁇ of the coenzyme binding domain.
  • NAD + The primary determinant of NAD + specificity is the presence of an aspartate residue, which forms double hydrogen bonds to both the 2'- and 3'-hydroxyl groups in the ribosyl moiety of NAD + and induces negative electrostatic potential to the binding site.
  • this residue in NADP + -dependent dehydrogenases is replaced by a smaller and uncharged residue such as Gly, Ala, and Ser, accompanied by the concurrent presence of an arginine residue that forms a positive binding pocket for the 2 '-phosphate group of NADP + .
  • Sorbitol dehydrogenases are typically NAD + -dependent, but there is one reported case (Wolfe et al., 1999) from silverleaf whitefly Bemisia argentifolii that is the only known enzyme showing a strict preference to NADP + , and a crystal structure of the enzyme is available (Protein Data Bank [PDB] ID:1E3J) (Banfield et al. 2001). Again, the homologous residues of the motif binding the signature phosphate moiety of NADP + has the characteristics of a smaller residue (serine) and positively charged residue (arginine) in the cofactor binding pocket. N. crassa LAD (ncLAD) shares homology with NADP + -dependent B. argentifolii SDH (43% identity, 61% positive).
  • dehydrogenase from Ghiconobacter oxydans (goXDH) was constructed that was able to use NADP + exclusively.
  • the goXDH is a short-chain xylitol dehydrogenase with a different fold and mechanism for catalysis than medium-chain sugar alcohol dehydrogenases (including ncLAD)
  • the mutations conferring cofactor specificity are of the same type, with amino acids aspartate and methionine mutated to a smaller residue serine and positively-charged arginine, respectively.
  • Example 2 Reversal of Cofactor Specificity of N. crassa LAD by Rational
  • Single mutants of LAD were generated as described in the Materials and Methods section, and consisted of mutating D211 to smaller amino acid residues (Ala, Gly, Ser, Thr, or Val), 1212 to a basic residue (Lys or Arg), and D213 to serine. All of these were expressed in E. coli and SDS-PAGE analysis was performed to determine the solubility of these mutant LADs. However, the solubility of these mutants was variable, with D21 IS, I212R, I212K and D213S being the most soluble, followed by D211 A and D21 1G at about half of the wild- type enzyme level.
  • D21 IT and D211 V were dramatically reduced compared to that of the wild type, although purification of these LADs from cell lysate did result in a small amount of recovered enzyme (-0.1-0.2 mg /g cells). All mutant enzymes were purified to homogeneity and then analyzed by HPLC to determine the effects of the mutations, and three single mutants D211 A, D21 1G, and D21 IS all showed improvements in NADP + activity.
  • ncLAD-AR, -GR, and - SR were created, expressed, and purified. HPLC analysis showed further improved activity towards NADP + , with the largest gains in activity being with ncLAD- AR and ncLAD-SR.
  • ncLAD-SRN displayed almost 1.4-fold higher k cat than parent ncLAD-SR, but suffered a 3-fold loss in affinity as the K m ⁇ ADP ⁇ ose from 0.48 mM for ncLAD-SR to 1.45 mM for ncLAD-SRN.
  • the overall catalytic efficiency decreased 2- fold.
  • the ncLAD-SRN mutant was also significantly less stable than the ncLAD-SR parent, with high amount of precipitated enzyme occurring during the buffer exchange and concentration steps of the purification protocol (Materials and Methods Section).
  • Example 3 Screening Procedure Validation and EP-PCR Results Before attempting to screen a mutant library, particularly a larger-size library generated from error-prone PCR (EP-PCR), it is particularly important to verify the reproducibility of the screening procedure (Salazar et al, 2009; Wang et al., 2006) Variability inherent in every screen arises from various factors such as varying cell growth rates, expression of enzyme from multiple-copy vectors, cell lysis efficiency, pipetting errors, etc. These variations must be minimized in order to eliminate false positives and simplify data interpretation.
  • NAD(PJH at 340 nm is commonly used to measure the activity of dehydrogenases. Therefore, a 96-well plate screening assay was developed for high- throughput analysis of a library of error-prone PCR mutants of ncLAD-SR.
  • E. coli BL21(DE3) cells harboring plasmid pET-28a ncLAD-SR were used to inoculate 96-well plates and following growth, induction, and lysing steps, the reproducibility of NADP + activity measurement was tested by calculating the coefficient of variance (CV). The assay was confirmed to be sufficient with a coefficient of variance (CV) of
  • ncLAD-SR Three different libraries of ncLAD-SR, created using 0.10, 0.15, and 0.20 mM Mn 2+ were tested with the validated screening procedure. Based on these results, an optimal concentration of 0.15 mM Mn 2+ resulted in 40-50% of the clones being inactive, which correlates to approximately 1-2 amino acid substitutions per gene.
  • ncLAD has 364 amino acids, so a library of this size can have 7,280 single amino acid substitutions.
  • Example 4 Kinetic Data of the Engineered ncLAD Mutants.
  • the kinetic parameters of the parent wild-type and engineered mutant ncLAD enzymes towards substrates NAD + , NADP + , and L-arabinitol were determined.
  • the best first round rational design generated mutant, D21 IS showed a dramatic decrease in activity towards NAD + , with minimal yet detectable activity increase towards NADP + .
  • the best second round rational design mutant, D21 IS/1212R displayed a significant reversal in cofactor specificity, although still had ⁇ 5-fold lower kcatlKm than the wild-type had with NAD .
  • Single mutants of LAD were generated as described in the Materials and Methods section, and consisted of mutating D211 to smaller amino acid residues (Ala, Gly, Ser, Thr, or Val), 1212 to a basic residue (Lys or Arg), and D213 to serine.
  • the best mutants (D211 A, D211 G, and D211 S) were then subj ected to site-directed mutagene si s for introduction of arginine at position 212, with improvements found in ncLAD-AR and ncLAD-SR. Targeting position 213 with replacement with serine did not improve activity and resulted in insoluble protein.
  • the double mutants provided a reproducible positive control in a 96-well plate formatted screening procedure, and saturation mutagenesis at position 213 was performed.
  • the best mutant found had a D213N mutation, but this enzyme was found to be less stable than previous rounds, with noticeable amounts of white precipitate forming during purification and concentration, leading to kinetic parameter analysis that did not match the screening results. Therefore, the best double mutant, ncLAD-SR, was used in an error-prone PCR library screen in an attempt to enhance NADP + -activity and improve solubility and stability.
  • the final mutant, ncLAD-3x contained the two rational design mutations (D211 A/1212R) and a third mutation S348T.
  • E. coli expression strain BL21(DE3) was transformed with this expression vector and single colonies of transformed E. coli were used to inoculate 5 ml of LB medium supplemented with kanamycin (50 mg/ml). Five hours following inoculation, the small-scale culture was added to 1 L of LB medium containing kanamycin (100 ⁇ g/ml) and 0.5 mM ZnCl 2 and grown at 37 °C.
  • Recombinant proteins were purified from the clarified supernatant by virtue of the amino terminal polyhistidine tag using a Talon resin (Clontech) column charged with cobalt chloride. Following elution from the cobalt affinity resin, the cleavable polyhistidine tag was removed using thrombin (GE Healthcare: 1 U/mg protein). The protein was further purified by anion exchange (5 ml HiTrap Q: GE Healthcare) and size exclusion chromatographies (Superdex 75 16/60: G. E. Healthcare) prior to
  • Crystals grew within a week and reached a maximum size of 0.2 mm x 0.4 mm x 0.4 mm. Crystals were briefly soaked in the precipitant solution supplemented with 25% glycerol, and vitrified by plunging directly into liquid nitrogen prior to data collection.
  • Diffraction data were collected to a limiting resolution of 2.6 A at an insertion device line (LS-CAT-Sector 21ID-D, Advanced Photon Source, Argonne, IL), and integrated and scaled using the HKL2000 package (Otwinowski et al., 2003).
  • Crystallographic phases were determined by the molecular replacement method (McCoy, 2007) using the refined coordinates of human sorbitol dehydrogenase (48% identity over 306 residues) (Johansson et al., 2001;
  • the plates were centrifuged at 4,000 rpm for 15 minutes and the media was decanted.
  • Cell pellets were lysed by resuspension in 100 ⁇ , lysis buffer consisting of 100 mM phosphate buffer (pH 7.0) with 1 mg/mL lysozyme.
  • Adhesive labels were placed on the 96-well plates and vortexed until cell pellets were uniformly resuspended, and then followed by two freeze-thawing steps.
  • 96-well plates containing cell lysates were then centrifuged at 4,000 rpm at 4 °C for 15 minutes, and 20 ⁇ L of cell lysates was transferred to a fresh 96-well plate.
  • Enzyme Kinetics The kinetic rate constants for the purified N-His 6 tagged wild- type ('Hise' disclosed as SEQ ID NO: 11) and mutant LAD enzymes were determined as described elsewhere (Sullivan and Zhao, 2007). Briefly, the initial rates of reaction were determined by monitoring the increase in absorbance of NAD(P)H at 340 nm. Initial rates were measured at 25 °C using a Cary UV 100 Bio UV-Vis spectrophotometer (Varian) in triplicate. Reactions were initiated by addition of—1-20 ⁇ g purified LAD enzyme with N-terminal His 6 tag ('Hiss' disclosed as SEQ ID NO: 11).
  • Enzyme concentrations were determined with the Bio-Rad Protein Assay (Bio-Rad) with bovine serum albumin (Sigma) as standard.
  • Bio-Rad Bio-Rad Protein Assay
  • bovine serum albumin Sigma
  • L-arabinitol or NAD(P) + concentrations were varied from below the K m value to at least -times higher than the K m value, with saturating concentrations of the other substrate/cofactor present.
  • the Michaelis-Menton equation was fitted to the data using the non-linear curve fitting with least squares regression analysis in OriginPro 8 (OriginLab Corporation) to determine k ca t and K m values.
  • LAD L-arabinitol 4-dehydrogenase
  • ncLAD Neurospora crassa
  • psXDH Pichia stipitis xylitol dehydrogenase
  • RMSD root mean square deviation
  • Bond angles (°) 0.013 Highest resolution shell is shown in parenthesis.
  • R-factor SflFofcsj-klFo b Dffi
  • ncLAD-SRN 5 ⁇ 100 714 1.45 496
  • ncLAD-3x > 5 ⁇ 100 1210 0.55 2190
  • AH assays run in triplicate at 25 °C in 50 mM Tris-HCl, pH 8.0
  • Table 2 discloses ncLAD-wt as SEQ ID NO: 1, ncLAD-S as SEQ ID NO: 2, ncLAD-SR as SEQ ID NO: 3, ncLAD-SRN as SEQ ID NO: 5, ncLAD-3x as SEQ ID NO: 4 and "His 6 " as SEQ ID NO: 11.
  • Table 3 discloses ncLAD-wt as SEQ ID NO: 1, ncLAD-S as SEQ ID NO: 2, ncLAD-SR as SEQ ID NO: 3, ncLAD-3x as SEQ ID NO: 4 and "His 6 " as SEQ ID NO: 1 1.

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Abstract

Selon l'invention, pour élucider les déterminants de reconnaissance du substrat et du cofacteur sur LAD, la première structure cristallographique aux rayons X à haute résolution d'un LAD a été déterminée à une résolution de 2,6. La structure cristalline du complexe ncLAD-NAD+ a été utilisée avec succès pour l'ingénierie biologique de variants à une spécificité de cofacteur modifiée vis-à-vis de NADP+. Ces résultats constituent le cadre de l'ingénierie biologique de variants avec une plus grande activité qui peuvent surmonter le goulot d'étranglement dans le catabolisme du pentose dans la levure.
PCT/US2011/032486 2010-04-15 2011-04-14 Mutants de la l-arabinitol 4-déshydrogénase provenant de neurospora crassa WO2011130509A1 (fr)

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Title
BAE ET AL.: "Structure and Engineering of L-Arabinitol 4-Dehydrogenase from Neurospora crassa", J. MOL. BIOL., vol. 402, no. 22, 22 July 2010 (2010-07-22), pages 230 - 240 *
KIM ET AL.: "Cloning, characterization, and engineering of fungal L-arabinitol dehydrogenases", APPL. MICROBIOL. BIOTECHNOL., vol. 87, 23 April 2010 (2010-04-23), pages 1407 - 1414 *
NAIR ET AL.: "Biochemical Characterization of an L-Xylulose Reductase from Neurospora crassa.", APPL AND ENVIRON,MICROBIOL, vol. 73, 2007, pages 2001 - 2004 *
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