Abstract:

Lysine270 has been found to be a key amino acid that forms the binding pocket of PsXR （Piehia stipitis xylose reduetase） with nieotinamide adenine dinucleo,Lide phosphate （NADPH） or with nieotinamide adenine dinucleotide （NADH）. In order to investigate the effect of Lysine270 on the PsXR coenzyme specificity, 19 XR mu- tants were produced by substituting 19 amino acids tot Lysine270, and the interaction between XR mutants and NAD^＋ or NADP ~ was assessed by means of homology modeling and molecular docking. Then, K270R and K270N mutants were chosen to perform the bioinformatic analysis. After being induced by isopropyhhio-β-d-galactoside （IPTG） in Escherichia coli （E. coli）, the xylose reduetases of wild type and mutagenesis were purified and finally used to investigate the enzymatic properties. The results show that   K270R mutagenesis reduces the binding capability of XR with NADP^＋ and results in an increase of Michaelis constant from 0. 025 mmol/L to 0. 050 mmol/L;   K270N mutagenesis makes XR bind with NAD^＋ only; and   the coenzyme dependence of rationally-designed K270N completely reverses from NADPH to NADH.

Key words: xylose reductase, site-directed mutagencsis, coenzyme specificity, bioinformation