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Literature summary for 1.1.1.9 extracted from
- 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 .
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+ | Rhizomucor pusillus |
| D205A/I206R | site-directed mutagenesis, coenzyme preference of the mutant RpXDH is reversed from NAD+ to NADP+ | Rhizomucor pusillus |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| AgNO3 | complete inhibition | Rhizomucor pusillus |  |
| CdCl2 | complete inhibition | Rhizomucor pusillus | |
| CuCl2 | strong inhibition | Rhizomucor pusillus | |
| CuSO4 | strong inhibition | Rhizomucor pusillus | |
| EDTA | complete inhibition, enzyme activity can be partially restored by addition of Zn2+, Ni2+, Co2+, Ag+, Fe3+, and Mn2+ | Rhizomucor pusillus | |
| furfural | weak inhibition | Rhizomucor pusillus | |
| HgCl2 | complete inhibition | Rhizomucor pusillus | |
| iodoacetamide | complete inhibition | Rhizomucor pusillus | |
| iodoacetic acid | complete inhibition | Rhizomucor pusillus | |
| K4[Fe(CN)6] | weak inhibition | Rhizomucor pusillus | |
| N-ethylmaleimide | complete inhibition | Rhizomucor pusillus | |
| o-phenanthroline | complete inhibition | Rhizomucor pusillus | |
| p-chloromercuribenzoic acid | complete inhibition | Rhizomucor pusillus | |
| phenylmethanesulfonyl fluoride | weak inhibition | Rhizomucor pusillus | |
| trypsin inhibitor T-9378 | weak inhibition | Rhizomucor pusillus | |
| trypsin-chymotrypsin inhibitor T-9777 | weak inhibition | Rhizomucor pusillus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.027 | - |
NAD+ | native enzyme, pH 9.0, 35°C | Rhizomucor pusillus | |
| 0.186 | - |
NAD+ | recombinant wild-type enzyme, pH 9.0, 35°C | Rhizomucor pusillus | |
| 7.83 | - |
xylitol | native enzyme, pH 9.0, 35°C, with NAD+ | Rhizomucor pusillus | |
| 10.1 | - |
xylitol | recombinant wild-type enzyme, pH 9.0, 35°C, with NAD+ | Rhizomucor pusillus | |
| 13.5 | - |
NAD+ | recombinant mutant D205A, pH 9.0, 35°C | Rhizomucor pusillus | |
| 31.5 | - |
D-sorbitol | native enzyme, pH 9.0, 35°C | Rhizomucor pusillus | |
| 32.37 | - |
NAD+ | recombinant wild-type enzyme, pH 9.0, 35°C | Rhizomucor pusillus | |
| 34.3 | - |
NAD+ | recombinant mutant D205A/I206R, pH 9.0, 35°C | Rhizomucor pusillus | |
| 66.7 | - |
xylitol | recombinant mutant D205A, pH 9.0, 35°C, with NAD+ | Rhizomucor pusillus | |
| 149 | - |
ribitol | native enzyme, pH 9.0, 35°C | Rhizomucor pusillus | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| additional information | LiCl, NaCl, MgCl2, MgSO4, ZnCl2, ZnCl4, SnCl2, NiCl2, BaCl2, and PbCl2 do not affect the enzyme activity at 1 mM | Rhizomucor pusillus | |
| 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 |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| xylitol + NAD+ | Rhizomucor pusillus | - |
D-xylulose + NADH + H+ | - |
r | |
| xylitol + NAD+ | Rhizomucor pusillus NBRC 4578 | - |
D-xylulose + NADH + H+ | - |
r | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Rhizomucor pusillus | S6BFC0 | - |
- |
| Rhizomucor pusillus NBRC 4578 | S6BFC0 | - |
- |
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. 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 | - |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 11.7 | - |
purified recombinant wild-type enzyme, pH 9.0, 35°C | Rhizomucor pusillus |
| 57.8 | - |
purified native enzyme, pH 9.0, 35°C | Rhizomucor pusillus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| D-sorbitol + NAD+ | 71.8% activity compared to xylitol | Rhizomucor pusillus | L-sorbose + NADH + H+ | - |
? | |
| D-sorbitol + NAD+ | 71.8% activity compared to xylitol | Rhizomucor pusillus NBRC 4578 | L-sorbose + NADH + H+ | - |
? | |
| D-xylulose + NADH + H+ | - |
Rhizomucor pusillus | xylitol + NAD+ | - |
r | |
| D-xylulose + NADH + H+ | - |
Rhizomucor pusillus NBRC 4578 | xylitol + NAD+ | - |
r | |
| ribitol + NAD+ | 60.1% activity compared to xylitol | Rhizomucor pusillus | D-ribulose + NADH + H+ | - |
? | |
| ribitol + NAD+ | 60.1% activity compared to xylitol | Rhizomucor pusillus NBRC 4578 | D-ribulose + NADH + H+ | - |
? | |
| xylitol + NAD+ | - |
Rhizomucor pusillus | D-xylulose + NADH + H+ | - |
r | |
| xylitol + NAD+ | best substrate | Rhizomucor pusillus | D-xylulose + NADH + H+ | - |
r | |
| xylitol + NAD+ | - |
Rhizomucor pusillus NBRC 4578 | D-xylulose + NADH + H+ | - |
r | |
| xylitol + NAD+ | best substrate | Rhizomucor pusillus NBRC 4578 | D-xylulose + NADH + H+ | - |
r | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homodimer | 2 * 39185, sequence calculation, 2 * 41000, native enzyme, SDS-PAGE | Rhizomucor pusillus |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| NAD+-dependent xylitol dehydrogenase | - |
Rhizomucor pusillus |
| RpXDH | - |
Rhizomucor pusillus |
| XYL2 | - |
Rhizomucor pusillus |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 60 | - |
- |
Rhizomucor pusillus |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 10 | 65 | activity range, inactivation above | Rhizomucor pusillus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 1.16 | - |
NAD+ | recombinant mutant D205A/I206R, pH 9.0, 35°C | Rhizomucor pusillus | |
| 2 | 3.7 | xylitol | recombinant mutant D205A/I206R, pH 9.0, 35°C, with NAD+ | Rhizomucor pusillus | |
| 5.18 | - |
NAD+ | recombinant mutant D205A, pH 9.0, 35°C | Rhizomucor pusillus | |
| 6.35 | - |
NAD+ | recombinant wild-type enzyme, pH 9.0, 35°C | Rhizomucor pusillus | |
| 11.1 | - |
xylitol | recombinant wild-type enzyme, pH 9.0, 35°C, with NAD+ | Rhizomucor pusillus | |
| 19.7 | - |
xylitol | recombinant mutant D205A, pH 9.0, 35°C, with NAD+ | Rhizomucor pusillus | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 9 | - |
- |
Rhizomucor pusillus |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 4.5 | 10.5 | activity range, profile overview | Rhizomucor pusillus |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| additional information | NADP+ is a poor cofactor giving 3.1% activity compared to NAD+ | Rhizomucor pusillus | |
| NAD+ | dependent on | 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 |
| metabolism | xylitol dehydrogenase catalyzes the second step of D-xylose metabolism | Rhizomucor pusillus |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.034 | - |
NAD+ | recombinant mutant D205A/I206R, pH 9.0, 35°C | Rhizomucor pusillus | |
| 0.38 | - |
NAD+ | recombinant mutant D205A, pH 9.0, 35°C | Rhizomucor pusillus | |
| 11.1 | - |
xylitol | recombinant wild-type enzyme, pH 9.0, 35°C, with NAD+ | Rhizomucor pusillus | |
| 19.7 | - |
xylitol | recombinant mutant D205A, pH 9.0, 35°C, with NAD+ | Rhizomucor pusillus | |
| 23.7 | - |
xylitol | recombinant mutant D205A/I206R, pH 9.0, 35°C, with NAD+ | Rhizomucor pusillus | |
| 32.37 | - |
NAD+ | recombinant wild-type enzyme, pH 9.0, 35°C | Rhizomucor pusillus | |
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