Reversal of coenzyme specificity and improvement of catalytic efficiency of Pichiastipitis xylose reductase by rational site-directed mutagenesis |
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Authors: | Qi-Kai Zeng Hong-Li Du Jing-Fang Wang Dong-Qing Wei Xiao-Ning Wang Yi-Xue Li Ying Lin |
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Affiliation: | (1) School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, China;(2) College of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai, 200240, China;(3) Bioinformatics Center, Key Laboratory of Systems Biology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai, 200031, China |
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Abstract: | A major problem when xylose is used for ethanol production is the intercellular redox imbalance arising from different coenzyme specificities of xylose reductase (XR) and xylitol dehydrogenase. The residue Lys21 in XR from Pichia stipitis was subjected to site-directed mutagenesis to alter its coenzyme specificity. The N272D mutant exhibited improved catalytic efficiency when NADH was the coenzyme. Both K21A and K21A/N272D preferred NADH to NADPH, their catalytic efficiencies for NADPH were almost zero. The catalytic efficiency of K21A/N272D for NADH was almost 9-fold and 2-fold that of K21A and the wild-type enzyme, respectively. Complete reversal of coenzyme specificity toward NADH and improved catalytic efficiency were achieved. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Qi-Kai Zeng, Hong-Li Du, Jing-Fang Wang have contributed equally to this work. |
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Keywords: | Catalytic efficiency Coenzyme specificity Site-directed mutagenesis Substitution simulation Xylose reductase |
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