Application | Comment | Organism |
---|---|---|
synthesis | D-xylose is the second most abundant renewable sugar in nature, and its fermentation to ethanol has great economical potential. Unfortunately, Saccharomyces cerevisiae, which has been optimized for ethanol production, cannot utilize xylose efficiently, while D-xylulose, an isomerization product of D-xylose, can be assimilated. A major strategy for constructing xylose-fermenting Saccharomyces cerevisiae is to introduce genes involved in xylose metabolism from other organisms. Xylose reductase and xylitol dehydrogenase (EC 1.1.1.9) from the xylose-fermenting yeast Pichia stipitis are cloned into Saccharomyces cerevisiae to allow xylose fermentation to ethanol. In this case, xylose is converted into xylulose by the sequential actions of two oxidoreductases. First, Pichia stipitis xylose reductase catalyses the reduction of xylose into xylitol with NAD(P)H as co-substrate. Xylitol is then oxidized by PsXDH (Pichia stipitis xylitol dehydrogenase) which uses NAD+ exclusively as co-substrate to yield xylulose. The different coenzyme specificity of the two enzymes xylose reductase and xylitol dehydrogenase, however, creates an intracellular redox imbalance, which results in low ethanol yields and considerable xylitol by-product formation. A mutant is constructed that shows an altered active site that is more unfavorable for NADPH than NADH in terms of both Km and kcat. There are potentials for application of the mutant (K270S/N272P/S271G/R276F) in constructing a more balanced xylose reductase/xylitol dehydrogenase pathway in recombinant xylose-fermenting Saccharomyces cerevisiae strains | Scheffersomyces stipitis |
Cloned (Comment) | Organism |
---|---|
expression of His-tagged enzyme in Escherichia coli | Scheffersomyces stipitis |
Protein Variants | Comment | Organism |
---|---|---|
K270S/N272P/S271G/R276F | the mutant shows a 25fold preference toward NADH over NADPH by a factor of about 13fold, or an improvement of about 42fold, as measured by the ratio of the specificity constant kcat/Km coenzyme. Compared with the wild-type, the kcat(NADH) is slightly lower, while the kcat(NADPH) decreases by a factor of about 10 | Scheffersomyces stipitis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0062 | - |
NADPH | pH 6.0, temperature not specified in the publication, wild-type enzyme, wild-type enzyme | Scheffersomyces stipitis | |
0.0106 | - |
NADH | pH 6.0, temperature not specified in the publication, wild-type enzyme, wild-type enzyme | Scheffersomyces stipitis | |
0.147 | - |
NADH | pH 6.0, temperature not specified in the publication, mutant enzyme K270S/N272P/S271G/R276F | Scheffersomyces stipitis | |
0.427 | - |
NADPH | pH 6.0, temperature not specified in the publication, mutant enzyme K270S/N272P/S271G/R276F | Scheffersomyces stipitis | |
82 | - |
D-xylose | pH 6.0, temperature not specified in the publication, cofactor: NADPH, wild-type enzyme | Scheffersomyces stipitis | |
90 | - |
D-xylose | pH 6.0, temperature not specified in the publication, cofactor: NADH, wild-type enzyme | Scheffersomyces stipitis | |
168 | - |
D-xylose | pH 6.0, temperature not specified in the publication, cofactor: NADPH, mutant enzyme K270S/N272P/S271G/R276F | Scheffersomyces stipitis | |
291 | - |
D-xylose | pH 6.0, temperature not specified in the publication, cofactor: NADH, mutant enzyme K270S/N272P/S271G/R276F | Scheffersomyces stipitis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-xylose + NADPH + H+ | Scheffersomyces stipitis | xylose reductase is one of the key enzymes for xylose fermentation | xylitol + NADP+ | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Scheffersomyces stipitis | - |
- |
- |
Purification (Comment) | Organism |
---|---|
- |
Scheffersomyces stipitis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-xylose + NADH + H+ | wild-type enzyme prefers NADPH over NADH | Scheffersomyces stipitis | xylitol + NAD+ | - |
? | |
D-xylose + NADPH + H+ | xylose reductase is one of the key enzymes for xylose fermentation | Scheffersomyces stipitis | xylitol + NADP+ | - |
? | |
D-xylose + NADPH + H+ | wild-type enzyme prefers NADPH over NADH | Scheffersomyces stipitis | xylitol + NADP+ | - |
? |
Synonyms | Comment | Organism |
---|---|---|
PsXR | - |
Scheffersomyces stipitis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.6 | - |
D-xylose | pH 6.0, temperature not specified in the publication, cofactor: NADPH, mutant enzyme K270S/N272P/S271G/R276F | Scheffersomyces stipitis | |
2.6 | - |
NADPH | pH 6.0, temperature not specified in the publication, mutant enzyme K270S/N272P/S271G/R276F | Scheffersomyces stipitis | |
12 | - |
D-xylose | pH 6.0, temperature not specified in the publication, cofactor: NADH, mutant enzyme K270S/N272P/S271G/R276F | Scheffersomyces stipitis | |
12 | - |
NADH | pH 6.0, temperature not specified in the publication, mutant enzyme K270S/N272P/S271G/R276F | Scheffersomyces stipitis | |
15.4 | - |
D-xylose | pH 6.0, temperature not specified in the publication, cofactor: NADH, wild-type enzyme | Scheffersomyces stipitis | |
15.4 | - |
NADH | pH 6.0, temperature not specified in the publication, wild-type enzyme, wild-type enzyme | Scheffersomyces stipitis | |
27.5 | - |
D-xylose | pH 6.0, temperature not specified in the publication, cofactor: NADPH, wild-type enzyme | Scheffersomyces stipitis | |
27.5 | - |
NADPH | pH 6.0, temperature not specified in the publication, wild-type enzyme, wild-type enzyme | Scheffersomyces stipitis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADH | wild-type enzyme prefers NADPH over NADH. Mutant enzyme K270S/N272P/S271G/R276F shows a 25fold preference toward NADH over NADPH by a factor of about 13fold, or an improvement of about 42fold, as measured by the ratio of the specificity constant kcat/Km coenzyme | Scheffersomyces stipitis | |
NADPH | wild-type enzyme prefers NADPH over NADH. Mutant enzyme K270S/N272P/S271G/R276F shows a 25fold preference toward NADH over NADPH by a factor of about 13fold, or an improvement of about 42fold, as measured by the ratio of the specificity constant kcat/Km coenzyme | Scheffersomyces stipitis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
6.2 | - |
D-xylose | pH 6.0, temperature not specified in the publication, cofactor: NADPH, mutant enzyme K270S/N272P/S271G/R276F | Scheffersomyces stipitis | |
6.2 | - |
NADPH | pH 6.0, temperature not specified in the publication, mutant enzyme K270S/N272P/S271G/R276F | Scheffersomyces stipitis | |
81.7 | - |
D-xylose | pH 6.0, temperature not specified in the publication, cofactor: NADH, mutant enzyme K270S/N272P/S271G/R276F | Scheffersomyces stipitis | |
81.7 | - |
NADH | pH 6.0, temperature not specified in the publication, mutant enzyme K270S/N272P/S271G/R276F | Scheffersomyces stipitis | |
1460 | - |
D-xylose | pH 6.0, temperature not specified in the publication, cofactor: NADH, wild-type enzyme | Scheffersomyces stipitis | |
1460 | - |
NADH | pH 6.0, temperature not specified in the publication, wild-type enzyme, wild-type enzyme | Scheffersomyces stipitis | |
4648 | - |
D-xylose | pH 6.0, temperature not specified in the publication, cofactor: NADPH, wild-type enzyme | Scheffersomyces stipitis | |
4648 | - |
NADPH | pH 6.0, temperature not specified in the publication, wild-type enzyme, wild-type enzyme | Scheffersomyces stipitis |