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Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc.
J Biol Chem. 2005 Mar 18; 280(11):10340-9.JB

Abstract

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(+).

Authors+Show Affiliations

Institute of Advanced Energy, Kyoto University, Gokasyo, Uji, Kyoto 611-0011, Japan.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15623532

Citation

Watanabe, Seiya, et al. "Complete Reversal of Coenzyme Specificity of Xylitol Dehydrogenase and Increase of Thermostability By the Introduction of Structural Zinc." The Journal of Biological Chemistry, vol. 280, no. 11, 2005, pp. 10340-9.
Watanabe S, Kodaki T, Makino K. Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc. J Biol Chem. 2005;280(11):10340-9.
Watanabe, S., Kodaki, T., & Makino, K. (2005). Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc. The Journal of Biological Chemistry, 280(11), 10340-9.
Watanabe S, Kodaki T, Makino K. Complete Reversal of Coenzyme Specificity of Xylitol Dehydrogenase and Increase of Thermostability By the Introduction of Structural Zinc. J Biol Chem. 2005 Mar 18;280(11):10340-9. PubMed PMID: 15623532.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc. AU - Watanabe,Seiya, AU - Kodaki,Tsutomu, AU - Makino,Keisuke, Y1 - 2004/12/28/ PY - 2004/12/30/pubmed PY - 2005/4/26/medline PY - 2004/12/30/entrez SP - 10340 EP - 9 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 280 IS - 11 N2 - 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(+). SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/15623532/Complete_reversal_of_coenzyme_specificity_of_xylitol_dehydrogenase_and_increase_of_thermostability_by_the_introduction_of_structural_zinc_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=15623532 DB - PRIME DP - Unbound Medicine ER -