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Literature summary extracted from

  • Hou, J.; Shen, Y.; Li, X.P.; Bao, X.M.
    Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae (2007), Lett. Appl. Microbiol., 45, 184-189.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.9 genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) are introduced into Saccharomyces cerevisiae, which already contain the Pichia stipitis XYL1 gene (encoding xylose reductase) and the endogenously overexpressed XKS1 gene (encoding xylulokinase) Scheffersomyces stipitis
1.1.1.10 expression of Pichia stipitis mutated XYL2 (D207/I208R/F209S or XYL2 S96C/S99C/Y102C/D207A/I208R/F209S) in Saccharomyces cerevisiae Scheffersomyces stipitis

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.9 D207A/I208R/F209S mutant bearing a reversal of coenzyme specificity from NAD+ to NADP+ is introduced into Saccharomyces cerevisiae. kcat/Km (NAD+): 181 l/min/mmol, kcat/Km (NADP+): 2790 l/min/mmol, Km (xylitol in the presence of NAD+): 31.1 mM, NADP+-dependent activity: 0.782 U/mg, NAD+-dependent activity: 0.271 U/mg. In xylose fermentation a large decrease in xylitol and glycerol yield is shown, while the xylose consumption and ethanol yield are decreased Scheffersomyces stipitis
1.1.1.9 S96C/S99C/Y102C/D207A/I208R/F209S mutant bearing a reversal of coenzyme specificity from NAD+ to NADP+ and additional zinc-binding site for thermostability, is introduced into Saccharomyces cerevisiae. kcat/Km (NADP+): 10700 l/min/mmol, Km (xylitol in the presence of NAD+): 111 mM , NADP+-dependent activity: 0.689 U/mg, NAD+-dependent activity: 0.136 U/mg. The xylose consumption and ethanol yield are decreased, and the xylitol yield is increased, because of low XDH activity Scheffersomyces stipitis
1.1.1.10 D207/I208R/F209S kcat/Km of mutant enzyme for NAD+ dropps 15fold compared with the native enzyme, kcat/Km for NADP+ increases up to 4100fold Scheffersomyces stipitis
1.1.1.10 S96C/S99C/Y102C/D207A/I208R/F209S mutation produces a further 4fold improvement in the kcat/Km for NADP+ compared to mutant enzyme D207/I208R/F209S Scheffersomyces stipitis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.9 21.7
-
 xylitol wild-type XDH: kcat/Km (NAD+): 2760 l/min/mmol, kcat/Km (NADP+): 2790 l/min/mmol Scheffersomyces stipitis
1.1.1.9 31.1
-
 xylitol mutant D207A/I208R/F209S: kcat/Km (NAD+): 181 l/min/mmol, kcat/Km (NADP+): 0.65 l/min/mmol Scheffersomyces stipitis
1.1.1.9 111
-
 xylitol mutant S96C/S99C/Y102C/D207A/I208R/F209S: kcat/Km (NADP+): 10700 l/min/mmol Scheffersomyces stipitis

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.9 Scheffersomyces stipitis
-
-
-
1.1.1.10 Scheffersomyces stipitis
-
-
-

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.1.1.9 additional information
-
 wild-type: NADP+-dependent activity: 0.005 U/mg, NAD+-dependent activity: 1.654 U/mg, mutant D207A/I208R/F209S: NADP+-dependent activity: 0.782 U/mg, NAD+-dependent activity: 0.271 U/mg, mutant S96C/S99C/Y102C/D207A/I208R/F209S: NADP+-dependent activity: 0.698 U/mg, NAD+-dependent activity: 0.136 U/mg Scheffersomyces stipitis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.9 xylitol + NAD+
-
 Scheffersomyces stipitis D-xylulose + NADH + H+
-
 ?
1.1.1.9 xylitol + NADP+
-
 Scheffersomyces stipitis D-xylulose + NADPH + H+
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.1.1.9 XDH
-
 Scheffersomyces stipitis
1.1.1.9 XL2
-
 Scheffersomyces stipitis
1.1.1.9 xylitol dehydrogenase
-
 Scheffersomyces stipitis