Site-directed mutagenesis of the cysteine residues in the Pichia stipitis xylose reductase.
Xylose reductase catalyzes the reduction of xylose to xylitol and is known to play a pivotal role in pentose metabolism in yeasts. We previously showed that a cystein residue may be involved in binding of the coenzyme NADPH to the Pichia stipitis xylose reductase through chemical modification studies. The question arose as to which of the three cysteine residues in this enzyme may be involved in coenzyme binding. We cloned the XYL1 gene encoding xylose reductase from P. stipitis into the phagemid pEMBL18(+) suitable for site-directed mutagenesis. Each of the three cysteine residues (Cys19, Cys27 and Cys130) was individually mutated to serine. All three Cys-->Ser variants remained functional, but with reduced catalytic activity. Sensitivity of the P. stipitis xylose reductase to thiol-specific reagents was attributed to both Cys27 and Cys130 residues as substitution of either residue with Ser resulted in a significant but incomplete loss of sensitivity to PCMBS. The apparent Km values of the Cys variants for NADPH, NADH and xylose did not differ from those of the wild-type enzyme isolated from yeast by more than 4-fold. Our results suggest that none of the Cys residues are directly involved in NADPH binding, although Cys130 may reside in or near the coenzyme binding region and might play a role in coenzyme specificity.
Zhang
Y
Y
Department of Environmental Biology, University of Guelph, Ontario, Canada.
Lee
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H
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000449:Aldehyde Reductase / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D003545:Cysteine / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D012694:Serine / Q000235:genetics; D014994:Xylose / Q000378:metabolism
D003067:Coenzymes; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009243:NAD; D012694:Serine; D009249:NADP; D002731:4-Chloromercuribenzenesulfonate; D014994:Xylose; D000449:Aldehyde Reductase; D003545:Cysteine