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Literature summary for 1.1.1.430 extracted from

  • Liang, L.; Zhang, J.; Lin, Z.
    Altering coenzyme specificity of Pichia stipitis xylose reductase by the semi-rational approach CASTing (2007), Microb. Cell Fact., 6, 0000 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.1.1.430

Application

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(Commentary)

Cloned (Comment) Organism
expression of His-tagged enzyme in Escherichia coli Scheffersomyces stipitis

Protein Variants

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Scheffersomyces stipitis
-
-
-

Purification (Commentary)

Purification (Comment) Organism
-
 Scheffersomyces stipitis

Substrates and Products (Substrate)

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

Synonyms Comment Organism
PsXR
-
 Scheffersomyces stipitis

Turnover Number [1/s]

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

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 [mM/s]

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