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

Metzger, Markus; Hollenberg, Cornelis P.

doi:10.1111/j.1432-1033.1995.0050o.x

Cited by 19 publications

(9 citation statements)

References 24 publications

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“…Comparison with Coenzyme Switching Studies for Oxidoreductases of the SDR and Other Families-Such a drastic change in coenzyme specificity by single mutation observed in the present study has not been reported yet for dehydrogenases of the SDR (37-39) and other families (17,19,40,41) in which systematic replacement of a set of amino acids in their ␤␣␤ folds is necessary to switch their coenzyme specificity. Although there has been no report on the mutation of the residue corresponding to Thr-38 of mouse lung CR in NADP(H)-dependent enzymes of the SDR family, the reverse-sense mutation, i.e.…”

Section: Fig 2 Effects Of Coenzymes On Thermal Inactivation Of Wt (supporting

confidence: 46%

Switch of Coenzyme Specificity of Mouse Lung Carbonyl Reductase by Substitution of Threonine 38 with Aspartic Acid

Nakanishi

Matsuura

Kaibe

et al. 1997

Journal of Biological Chemistry

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Mouse lung carbonyl reductase, a member of the short-chain dehydrogenase/reductase (SDR) family, exhibits coenzyme specificity for NADP(H) over NAD(H). Crystal structure of the enzyme-NADPH complex shows that Thr-38 interacts with the 2'-phosphate of NADPH and occupies the position spatially similar to an Asp residue of the NAD(H)-dependent SDRs that hydrogen-bonds to the hydroxyl groups of the adenine ribose of the coenzymes. Using site-directed mutagenesis, we constructed a mutant mouse lung carbonyl reductase in which Thr-38 was replaced by Asp (T38D), and we compared kinetic properties of the mutant and wild-type enzymes in both forward and reverse reactions. The mutation resulted in increases of more than 200-fold in the Km values for NADP(H) and decreases of more than 7-fold in those for NAD(H), but few changes in the Km values for substrates or in the kcat values of the reactions. NAD(H) provided maximal protection against thermal and urea denaturation of T38D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the single mutation converted the coenzyme specificity from NADP(H) to NAD(H). Calculation of free energy changes showed that the 2'-phosphate of NADP(H) contributes to its interaction with the wild-type enzyme. Changing Thr-38 to Asp destabilized the binding energies of NADP(H) by 3.9-4.5 kcal/mol and stabilized those of NAD(H) by 1.2-1.4 kcal/mol. These results indicate a significant role of Thr-38 in NADP(H) binding for the mouse lung enzyme and provide further evidence for the key role of Asp at this position in NAD(H) specificity of the SDR family proteins.

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Section: Fig 2 Effects Of Coenzymes On Thermal Inactivation Of Wt (supporting

confidence: 46%

Switch of Coenzyme Specificity of Mouse Lung Carbonyl Reductase by Substitution of Threonine 38 with Aspartic Acid

Nakanishi

Matsuura

Kaibe

et al. 1997

Journal of Biological Chemistry

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“…Similarly, attempts were made to increase the affinity of XDH for NADP + which resulted in improved ethanol production from xylose (Watanabe et al, 2007b;. The XYL2 gene has also been engineered by introduction of an NADP + recognition sequence from Thermoanaerobium brockii, resulting in an enzyme with equal K m values for NAD + and NADP + (Metzger and Hollenberg, 1995). The strain with the mutated XYL2 mediated growth on xylose when XYL1…”

Section: C H O +5adp+5pmentioning

confidence: 98%

Bioconversion of lignocellulose-derived sugars to ethanol by engineeredSaccharomyces cerevisiae

Madhavan

Srivastava

Kondo

et al. 2011

Critical Reviews in Biotechnology

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Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.

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“…This increased ethanol yield to 0AE4 g g )1 xylose and decreased xylitol production. In another attempt to recycle coenzyme by increasing the affinity of XDH for NADP + , an NADP + recognition sequence from Thermoanaerobium brockii was introduced into the XYL2 gene, resulting in equal apparent K m values for NAD + and NADP + (Metzger and Hollenberg 1995). Ehrensberger et al (2006) constructed a double mutant (D38S ⁄ M39R) of Gluconobacter oxydans XDH that was able to exclusively use NADP + , with no loss of activity.…”

mentioning

confidence: 99%

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

Hou

Shen

et al. 2007

Lett Appl Microbiol

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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 0·782 and 0·698 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.

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

Cited by 19 publications

References 24 publications

Switch of Coenzyme Specificity of Mouse Lung Carbonyl Reductase by Substitution of Threonine 38 with Aspartic Acid

Switch of Coenzyme Specificity of Mouse Lung Carbonyl Reductase by Substitution of Threonine 38 with Aspartic Acid

Bioconversion of lignocellulose-derived sugars to ethanol by engineeredSaccharomyces cerevisiae

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

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