Amino Acid Substitutions in the Yeast Pichia Stipitis Xylitol Dehydrogenase Coenzyme-Binding Domain Affect the Coenzyme Specificity

Abstract

Directed mutagenesis has been used to identify a set of amino acids in the Pichia stipitis xylitol dehydrogenase, encoded by the xylitol dehydrogenase gene XYL2, which is involved in specific NAD binding. Within the binding domain, a characteristic beta alpha beta-fold is centered around a glycine motif GXGXXG also containing conserved aspartate and lysine/arginine residues. The mutation D207-->G and the double mutation D207-->G and D210-->G increased the apparent Km for NAD ninefold and decreased the xylitol dehydrogenase activity to 47% and 35%, respectively, as compared to the unaltered enzyme. The introduction of the potential NADP-recognition sequence (GSRPVC) of the alcohol dehydrogenase from Thermoanaerobium brockii into the xylitol dehydrogenase allowed the mutant enzyme to use both NAD and NADP as cofactor with equal apparent Km values. Although this mutant enzyme displayed an unaltered NADP acceptance, the reduction of the NAD specificity in the stably expressed enzyme variant is an important first step towards the long-term goal to reverse the coenzyme specificity from NAD to NADP. The mutagenized XYL2 gene could still mediate growth of Saccharomyces cerevisiae transformants on xylose minimal-medium plates when expressed together with the xylose reductase gene (XYL1).

The expression of aPichia stipitis xylose reductase mutant with higherKM for NADPH increases ethanol production from xylose in recombinantSaccharomyces cerevisiae

“…Attempts have also been made to increase the affinity of XDH for the cofactor NADP þ . An NADP þ recognition sequence from Thermoanaerobium brockii was introduced in the XYL2 gene resulting in equal apparent K M values for NAD þ and NADP þ (Metzger and Hollenberg, 1995). The mutated XYL2 gene mediated xylose growth when coexpressed with the XYL1 gene in S. cerevisiae, however, ethanolic fermentation of xylose was not reported (Metzger and Hollenberg, 1995).…”

Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis

“…Therefore, modifying the coenzyme specificity of XR and/or XDH by protein engineering is one of the attractive challenges for achieving efficient ethanol fermentation from xylose using S. cerevisiae. In the case of XDH, Metzger & Hollenberg (1995) first attempted to identify a set of amino acid residues in PsXDH responsible for specificity to NAD + . They introduced the potential NADP + (H)-recognition sequence of Escherichia coli glutathione reductase (no homology with PsXDH) and thermophilic alcohol dehydrogenase (~30 % homology) into the homologous sequence in PsXDH.…”

Cloning and Overexpression of the Xylitol Dehydrogenase Gene from Bacillus pallidus and Its Application to L-Xylulose Production

“…[1][2][3][4][5][6][7] Erwinia uredovora (later renamed Pantoea ananatis) is the only bacterium known to have an activity related to L-xylulose formation from xylitol. Many studies have been conducted on the mechanisms and properties of the enzyme in this organism.…”