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Literature summary 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 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.9 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
1.1.1.B64 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

EC Number Protein Variants Comment Organism
1.1.1.9 D205A site-directed mutagenesis, coenzyme preference of the mutant RpXDH is partially reversed from NAD+ to NADP+ Rhizomucor pusillus
1.1.1.9 D205A/I206R site-directed mutagenesis, coenzyme preference of the mutant RpXDH is reversed from NAD+ to NADP+ Rhizomucor pusillus
1.1.1.B64 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
1.1.1.B64 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

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.1.1.9 AgNO3 complete inhibition Rhizomucor pusillus
1.1.1.9 CdCl2 complete inhibition Rhizomucor pusillus
1.1.1.9 CuCl2 strong inhibition Rhizomucor pusillus
1.1.1.9 CuSO4 strong inhibition Rhizomucor pusillus
1.1.1.9 EDTA complete inhibition, enzyme activity can be partially restored by addition of Zn2+, Ni2+, Co2+, Ag+, Fe3+, and Mn2+ Rhizomucor pusillus
1.1.1.9 furfural weak inhibition Rhizomucor pusillus
1.1.1.9 HgCl2 complete inhibition Rhizomucor pusillus
1.1.1.9 iodoacetamide complete inhibition Rhizomucor pusillus
1.1.1.9 iodoacetic acid complete inhibition Rhizomucor pusillus
1.1.1.9 K4[Fe(CN)6] weak inhibition Rhizomucor pusillus
1.1.1.9 N-ethylmaleimide complete inhibition Rhizomucor pusillus
1.1.1.9 o-phenanthroline complete inhibition Rhizomucor pusillus
1.1.1.9 p-chloromercuribenzoic acid complete inhibition Rhizomucor pusillus
1.1.1.9 phenylmethanesulfonyl fluoride weak inhibition Rhizomucor pusillus
1.1.1.9 trypsin inhibitor T-9378 weak inhibition Rhizomucor pusillus
1.1.1.9 trypsin-chymotrypsin inhibitor T-9777 weak inhibition Rhizomucor pusillus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.9 0.027
-
 NAD+ native enzyme, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.9 0.186
-
 NAD+ recombinant wild-type enzyme, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.9 7.83
-
 xylitol native enzyme, pH 9.0, 35°C, with NAD+ Rhizomucor pusillus
1.1.1.9 10.1
-
 xylitol recombinant wild-type enzyme, pH 9.0, 35°C, with NAD+ Rhizomucor pusillus
1.1.1.9 13.5
-
 NAD+ recombinant mutant D205A, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.9 31.5
-
 D-sorbitol native enzyme, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.9 32.37
-
 NAD+ recombinant wild-type enzyme, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.9 34.3
-
 NAD+ recombinant mutant D205A/I206R, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.9 66.7
-
 xylitol recombinant mutant D205A, pH 9.0, 35°C, with NAD+ Rhizomucor pusillus
1.1.1.9 149
-
 ribitol native enzyme, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.B64 0.712
-
 NADP+ recombinant mutant D205A/I206R, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.B64 2.54
-
 NADP+ recombinant mutant D205A, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.B64 9.2
-
 NADP+ native wild-type enzyme, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.B64 10.2
-
 NADP+ recombinant wild-type enzyme, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.B64 130
-
 xylitol recombinant mutant D205A/I206R, pH 9.0, 35°C, with NADP+ Rhizomucor pusillus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.1.1.9 additional information LiCl, NaCl, MgCl2, MgSO4, ZnCl2, ZnCl4, SnCl2, NiCl2, BaCl2, and PbCl2 do not affect the enzyme activity at 1 mM Rhizomucor pusillus
1.1.1.9 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
1.1.1.B64 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]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.1.1.9 87000
-
 native enzyme, gel filtration Rhizomucor pusillus
1.1.1.B64 87000
-
 native enzyme, gel filtration Rhizomucor pusillus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.9 xylitol + NAD+ Rhizomucor pusillus
-
 D-xylulose + NADH + H+
-
 r
1.1.1.9 xylitol + NAD+ Rhizomucor pusillus NBRC 4578
-
 D-xylulose + NADH + H+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.9 Rhizomucor pusillus S6BFC0
-
-
1.1.1.9 Rhizomucor pusillus NBRC 4578 S6BFC0
-
-
1.1.1.B64 Rhizomucor pusillus
-
 enzyme mutants D205A/I206R and D205A
-
1.1.1.B64 Rhizomucor pusillus NBRC 4578
-
 enzyme mutants D205A/I206R and D205A
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.9 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. 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
1.1.1.B64 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

EC Number Source Tissue Comment Organism Textmining
1.1.1.9 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
-
1.1.1.B64 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
-

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 11.7
-
 purified recombinant wild-type enzyme, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.9 57.8
-
 purified native enzyme, pH 9.0, 35°C Rhizomucor pusillus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.9 D-sorbitol + NAD+ 71.8% activity compared to xylitol Rhizomucor pusillus L-sorbose + NADH + H+
-
 ?
1.1.1.9 D-sorbitol + NAD+ 71.8% activity compared to xylitol Rhizomucor pusillus NBRC 4578 L-sorbose + NADH + H+
-
 ?
1.1.1.9 D-xylulose + NADH + H+
-
 Rhizomucor pusillus xylitol + NAD+
-
 r
1.1.1.9 D-xylulose + NADH + H+
-
 Rhizomucor pusillus NBRC 4578 xylitol + NAD+
-
 r
1.1.1.9 ribitol + NAD+ 60.1% activity compared to xylitol Rhizomucor pusillus D-ribulose + NADH + H+
-
 ?
1.1.1.9 ribitol + NAD+ 60.1% activity compared to xylitol Rhizomucor pusillus NBRC 4578 D-ribulose + NADH + H+
-
 ?
1.1.1.9 xylitol + NAD+
-
 Rhizomucor pusillus D-xylulose + NADH + H+
-
 r
1.1.1.9 xylitol + NAD+ best substrate Rhizomucor pusillus D-xylulose + NADH + H+
-
 r
1.1.1.9 xylitol + NAD+
-
 Rhizomucor pusillus NBRC 4578 D-xylulose + NADH + H+
-
 r
1.1.1.9 xylitol + NAD+ best substrate Rhizomucor pusillus NBRC 4578 D-xylulose + NADH + H+
-
 r
1.1.1.B64 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+
-
 ?
1.1.1.B64 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

EC Number Subunits Comment Organism
1.1.1.9 homodimer 2 * 39185, sequence calculation, 2 * 41000, native enzyme, SDS-PAGE Rhizomucor pusillus
1.1.1.B64 homodimer 2 * 39185, sequence calculation, 2 * 41000, native enzyme, SDS-PAGE Rhizomucor pusillus

Synonyms

EC Number Synonyms Comment Organism
1.1.1.9 NAD+-dependent xylitol dehydrogenase
-
 Rhizomucor pusillus
1.1.1.9 RpXDH
-
 Rhizomucor pusillus
1.1.1.9 XYL2
-
 Rhizomucor pusillus
1.1.1.B64 NADP+-dependent xylitol dehydrogenase
-
 Rhizomucor pusillus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.9 60
-
-
 Rhizomucor pusillus
1.1.1.B64 35
-
 assay at Rhizomucor pusillus

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
1.1.1.9 10 65 activity range, inactivation above Rhizomucor pusillus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.9 1.16
-
 NAD+ recombinant mutant D205A/I206R, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.9 2 3.7 xylitol recombinant mutant D205A/I206R, pH 9.0, 35°C, with NAD+ Rhizomucor pusillus
1.1.1.9 5.18
-
 NAD+ recombinant mutant D205A, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.9 6.35
-
 NAD+ recombinant wild-type enzyme, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.9 11.1
-
 xylitol recombinant wild-type enzyme, pH 9.0, 35°C, with NAD+ Rhizomucor pusillus
1.1.1.9 19.7
-
 xylitol recombinant mutant D205A, pH 9.0, 35°C, with NAD+ Rhizomucor pusillus
1.1.1.B64 2 3.7 NADP+ recombinant mutant D205A/I206R, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.B64 11.1
-
 NADP+ recombinant wild-type enzyme, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.B64 19.7
-
 NADP+ recombinant mutant D205A, pH 9.0, 35°C Rhizomucor pusillus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.9 9
-
-
 Rhizomucor pusillus
1.1.1.B64 9
-
 assay at Rhizomucor pusillus

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.1.1.9 4.5 10.5 activity range, profile overview Rhizomucor pusillus

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.9 additional information NADP+ is a poor cofactor giving 3.1% activity compared to NAD+ Rhizomucor pusillus
1.1.1.9 NAD+ dependent on Rhizomucor pusillus
1.1.1.B64 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

EC Number Organism Comment Expression
1.1.1.9 Rhizomucor pusillus D-xylose induces the enzyme expression leading to accumulation of xylitol up
1.1.1.B64 Rhizomucor pusillus D-xylose induces the enzyme expression leading to accumulation of xylitol up

General Information

EC Number General Information Comment Organism
1.1.1.9 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
1.1.1.9 metabolism xylitol dehydrogenase catalyzes the second step of D-xylose metabolism Rhizomucor pusillus
1.1.1.B64 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.9 0.034
-
 NAD+ recombinant mutant D205A/I206R, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.9 0.38
-
 NAD+ recombinant mutant D205A, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.9 11.1
-
 xylitol recombinant wild-type enzyme, pH 9.0, 35°C, with NAD+ Rhizomucor pusillus
1.1.1.9 19.7
-
 xylitol recombinant mutant D205A, pH 9.0, 35°C, with NAD+ Rhizomucor pusillus
1.1.1.9 23.7
-
 xylitol recombinant mutant D205A/I206R, pH 9.0, 35°C, with NAD+ Rhizomucor pusillus
1.1.1.9 32.37
-
 NAD+ recombinant wild-type enzyme, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.B64 0.0178
-
 NADP+ recombinant wild-type enzyme, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.B64 0.13
-
 NADP+ recombinant mutant D205A, pH 9.0, 35°C Rhizomucor pusillus
1.1.1.B64 0.557
-
 NADP+ recombinant mutant D205A/I206R, pH 9.0, 35°C Rhizomucor pusillus