Xylitol Dehydrogenase (xylitol + dehydrogenase)

Distribution by Scientific Domains


Selected Abstracts


The metabolic role and evolution of l -arabinitol 4-dehydrogenase of Hypocrea jecorina

FEBS JOURNAL, Issue 10 2004
Manuela Pail
l -Arabinitol 4-dehydrogenase (Lad1) of the cellulolytic and hemicellulolytic fungus Hypocrea jecorina (anamorph: Trichoderma reesei) has been implicated in the catabolism of l -arabinose, and genetic evidence also shows that it is involved in the catabolism of d -xylose in xylitol dehydrogenase (xdh1) mutants and of d -galactose in galactokinase (gal1) mutants of H. jecorina. In order to identify the substrate specificity of Lad1, we have recombinantly produced the enzyme in Escherichia coli and purified it to physical homogeneity. The resulting enzyme preparation catalyzed the oxidation of pentitols (l -arabinitol) and hexitols (d -allitol, d -sorbitol, l -iditol, l -mannitol) to the same corresponding ketoses as mammalian sorbitol dehydrogenase (SDH), albeit with different catalytic efficacies, showing highest kcat/Km for l -arabinitol. However, it oxidized galactitol and d -talitol at C4 exclusively, yielding l -xylo-3-hexulose and d -arabino-3-hexulose, respectively. Phylogenetic analysis of Lad1 showed that it is a member of a terminal clade of putative fungal arabinitol dehydrogenase orthologues which separated during evolution of SDHs. Juxtapositioning of the Lad1 3D structure over that of SDH revealed major amino acid exchanges at topologies flanking the binding pocket for d -sorbitol. A lad1 gene disruptant was almost unable to grow on l -arabinose, grew extremely weakly on l -arabinitol, d -talitol and galactitol, showed reduced growth on d -sorbitol and d -galactose and a slightly reduced growth on d -glucose. The weak growth on l -arabinitol was completely eliminated in a mutant in which the xdh1 gene had also been disrupted. These data show not only that Lad1 is indeed essential for the catabolism of l -arabinose, but also that it constitutes an essential step in the catabolism of several hexoses; this emphasizes the importance of such reductive pathways of catabolism in fungi. [source]


Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054

FEMS YEAST RESEARCH, Issue 3 2003
C.Fredrik Wahlbom
Abstract The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding d -xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on d -xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only d -xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h,1 for S. cerevisiae TMB 3399 to 0.14 h,1 for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h,1. All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose,1 and 0.001, 0.10, and 0.16 g ethanol g biomass,1 h,1 for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein,1, was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant. [source]


Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

LETTERS IN APPLIED MICROBIOLOGY, Issue 2 2007
J. Hou
Abstract Aims:, To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae. Methods and Results:, The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP+ -dependent activity in both strains with mutated XDH, reaching 0782 and 0698 U mg,1. In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity. Conclusions:, Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation. Significance and Impact of the Study:, The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important. [source]