Literature summary for 1.1.1.B64 extracted from

Yamasaki-Yashiki, S.; Komeda, H.; Hoshino, K.; Asano, Y.
Molecular analysis of NAD+-dependent xylitol dehydrogenase from the zygomycetous fungus Rhizomucor pusillus and reversal of the coenzyme preference (2014), Biosci. Biotechnol. Biochem., 78, 1943-1953 .

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Cloned(Commentary)

Cloned (Comment)	Organism
gene xyl2, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Rhizomucor pusillus

Protein Variants

Protein Variants	Comment	Organism
D205A	site-directed mutagenesis, coenzyme preference of the mutant RpXDH is partially reversed from NAD+ to NADP+, cf. EC 1.1.1.9	Rhizomucor pusillus
D205A/I206R	site-directed mutagenesis, coenzyme preference of the mutant RpXDH is reversed from NAD+ to NADP+, cf. EC 1.1.1.9	Rhizomucor pusillus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.712	-	NADP+	recombinant mutant D205A/I206R, pH 9.0, 35°C	Rhizomucor pusillus
2.54	-	NADP+	recombinant mutant D205A, pH 9.0, 35°C	Rhizomucor pusillus
9.2	-	NADP+	native wild-type enzyme, pH 9.0, 35°C	Rhizomucor pusillus
10.2	-	NADP+	recombinant wild-type enzyme, pH 9.0, 35°C	Rhizomucor pusillus
130	-	xylitol	recombinant mutant D205A/I206R, pH 9.0, 35°C, with NADP+	Rhizomucor pusillus

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Zn2+	required, the conserved coenzyme binding motif (GxGxxG) and zinc-ADH signature (GHExxGxxxxxGxxV) are observed in the amino acid sequence of RpXDH at position 181-186 and 70-84. The ligand binding residues for catalytic zinc (residue C46, H71, E72, and E157) and structural zinc (residue C101, C104, C107, and C115) are found in the RpXDH sequence	Rhizomucor pusillus

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
87000	-	native enzyme, gel filtration	Rhizomucor pusillus

Organism

Organism	UniProt	Comment	Textmining
Rhizomucor pusillus	-	enzyme mutants D205A/I206R and D205A	-
Rhizomucor pusillus NBRC 4578	-	enzyme mutants D205A/I206R and D205A	-

Purification (Commentary)

Purification (Comment)	Organism
native enzyme from Rhizomucor pusillus strain NBRC 4578 by anion exchange chromatography, ammonium sulfate fractionation, and hydrophobic interaction chromatography, followed by ultrafiltration, and a another different step of anion exchange chromatography, ultrafiltration, and gel filtration. The next purification steps are Reactive Red 120 affinity chromatography, dialysis, and ultrafiltration. Recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Rhizomucor pusillus

Purification (Comment)

Organism

native enzyme from Rhizomucor pusillus strain NBRC 4578 by anion exchange chromatography, ammonium sulfate fractionation, and hydrophobic interaction chromatography, followed by ultrafiltration, and a another different step of anion exchange chromatography, ultrafiltration, and gel filtration. The next purification steps are Reactive Red 120 affinity chromatography, dialysis, and ultrafiltration. Recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography

Rhizomucor pusillus

Source Tissue

Source Tissue	Comment	Organism	Textmining
additional information	expression of xyl2 gene is increased during exponential growth phase and maintains in stationary phase at 96 h in the D-xylose culture	Rhizomucor pusillus	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
xylitol + NADP+	substrate of enzyme mutants D205A and D205A/I206R, very low activity with the wild-type enzyme, which highly prefers NAD+, cf. EC 1.1.1.9	Rhizomucor pusillus	D-xylulose + NADPH + H+	-	?
xylitol + NADP+	substrate of enzyme mutants D205A and D205A/I206R, very low activity with the wild-type enzyme, which highly prefers NAD+, cf. EC 1.1.1.9	Rhizomucor pusillus NBRC 4578	D-xylulose + NADPH + H+	-	?

Subunits

Subunits	Comment	Organism
homodimer	2 * 39185, sequence calculation, 2 * 41000, native enzyme, SDS-PAGE	Rhizomucor pusillus

Synonyms

Synonyms	Comment	Organism
NADP+-dependent xylitol dehydrogenase	-	Rhizomucor pusillus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
35	-	assay at	Rhizomucor pusillus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
2	3.7	NADP+	recombinant mutant D205A/I206R, pH 9.0, 35°C	Rhizomucor pusillus
11.1	-	NADP+	recombinant wild-type enzyme, pH 9.0, 35°C	Rhizomucor pusillus
19.7	-	NADP+	recombinant mutant D205A, pH 9.0, 35°C	Rhizomucor pusillus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
9	-	assay at	Rhizomucor pusillus

Cofactor

Cofactor	Comment	Organism	Structure
NADP+	cofactor of enzyme mutants D205A and D205A/I206R, very low activity (3.1%) with the wild-type enzyme, which highly prefers NAD+ (100%), cf. EC 1.1.1.9	Rhizomucor pusillus

Expression

Organism	Comment	Expression
Rhizomucor pusillus	D-xylose induces the enzyme expression leading to accumulation of xylitol	up

General Information

General Information	Comment	Organism
evolution	the conserved coenzyme binding motif (GxGxxG) and zinc-ADH signature (GHExxGxxxxxGxxV) are observed in the amino acid sequence of RpXDH at position 181-186 and 70-84 and are completely conserved among RpXDH, XDHs, and SDHs from other filamentous fungi and yeasts	Rhizomucor pusillus

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.0178	-	NADP+	recombinant wild-type enzyme, pH 9.0, 35°C	Rhizomucor pusillus
0.13	-	NADP+	recombinant mutant D205A, pH 9.0, 35°C	Rhizomucor pusillus
0.557	-	NADP+	recombinant mutant D205A/I206R, pH 9.0, 35°C	Rhizomucor pusillus