Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc.
Pichia stipitis NAD(+)-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD(+) to NADP(+) and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp(207), Ile(208), Phe(209), and Asn(211) in the discrimination between NAD(+) and NADP(+). Single mutants (D207A, I208R, F209S, and N211R) improved 5 approximately 48-fold in catalytic efficiency (k(cat)/K(m)) with NADP(+) compared with the wild type but retained substantial activity with NAD(+). The double mutants (D207A/I208R and D207A/F209S) improved by 3 orders of magnitude in k(cat)/K(m) with NADP(+), but they still preferred NAD(+) to NADP(+). The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed more than 4500-fold higher values in k(cat)/K(m) with NADP(+) than the wild-type enzyme, reaching values comparable with k(cat)/K(m) with NAD(+) of the wild-type enzyme. Because most NADP(+)-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP(+).
Watanabe
Seiya
S
Institute of Advanced Energy, Kyoto University, Gokasyo, Uji, Kyoto 611-0011, Japan.
Kodaki
Tsutomu
T
Makino
Keisuke
K
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002384:Catalysis; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D050542:D-Xylulose Reductase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D000431:Ethanol / Q000737:chemistry; D005285:Fermentation; D006639:Histidine / Q000737:chemistry; D006358:Hot Temperature; D015151:Immunoblotting; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009243:NAD / Q000737:chemistry; D009249:NADP / Q000737:chemistry; D010843:Pichia / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors; D014994:Xylose / Q000737:chemistry; D015032:Zinc / Q000737:chemistry*
D008024:Ligands; D011994:Recombinant Proteins; D009243:NAD; D000431:Ethanol; D006639:Histidine; D009249:NADP; D014994:Xylose; D013401:Sugar Alcohol Dehydrogenases; D050542:D-Xylulose Reductase; D015032:Zinc; D003545:Cysteine