Cloned (Comment) | Organism |
---|---|
gene hspB, phylogenetic analysis and tree, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3) | Pseudomonas putida |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | stopped-flow spectroscopy and kinetic analysis | Pseudomonas putida | |
0.0294 | - |
NADH | pH 8.0, 25°C, recombinant His-tagged enzyme | Pseudomonas putida | |
0.173 | - |
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate | pH 8.0, 25°C, recombinant His-tagged enzyme | Pseudomonas putida |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2 | Pseudomonas putida | - |
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O | - |
? | |
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2 | Pseudomonas putida S16 | - |
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | - |
- |
- |
Pseudomonas putida S16 | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Pseudomonas putida |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2 = 2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O | catalytic mechanism, overview. In contrast to conclusions reported previously, the second product of the HspB reaction is shown to be succinate, with isotope labeling experiments providing direct evidence that the newly introduced oxygen atom of succinate is derived from H2O. Reduced HspB reacts with oxygen to form a C(4a)-(hydro)peroxyflavin intermediate before it is converted to the oxidized flavoenzyme species. The formed C(4a)-hydroperoxyflavin intermediate reacts with HSP to form an intermediate that is hydrolyzed to the products 2,5-dihydroxypyridine and succinate | Pseudomonas putida |
Storage Stability | Organism |
---|---|
-80°C, purified recombinant His-tagged enzyme HspB, as a frozen solution or dry powder, no change in activity for at least 3 months, pH 8.0 | Pseudomonas putida |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2 | - |
Pseudomonas putida | 2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O | - |
? | |
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2 | - |
Pseudomonas putida S16 | 2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O | - |
? | |
additional information | mass spectrometric analysis of substrates and products, with recombinant enzyme | Pseudomonas putida | ? | - |
? | |
additional information | mass spectrometric analysis of substrates and products, with recombinant enzyme | Pseudomonas putida S16 | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
6-hydroxy-3-succinoyl-pyridine 3-monooxygenase | - |
Pseudomonas putida |
HSP 3-monooxygenase | - |
Pseudomonas putida |
hspB | - |
Pseudomonas putida |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Pseudomonas putida |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
5.49 | - |
NADH | pH 8.0, 25°C, recombinant His-tagged enzyme | Pseudomonas putida | |
7.74 | - |
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate | pH 8.0, 25°C, recombinant His-tagged enzyme | Pseudomonas putida |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Pseudomonas putida |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Pseudomonas putida | |
NADH | - |
Pseudomonas putida |
General Information | Comment | Organism |
---|---|---|
evolution | phylogenetic analysis reveals that HspB is the most closely related to two p-nitrophenol 4-monooxygenases, and the experimental results exhibit that p-nitrophenol is a substrate of HspB | Pseudomonas putida |
additional information | free H2O2 does not catalyze the HspB enzyme reaction | Pseudomonas putida |
physiological function | 6-hydroxy-3-succinoyl-pyridine (HSP) 3-monooxygenase (HspB) is a flavoprotein essential to the pyrrolidine pathway of nicotine degradation, it catalyzes pyridine-ring beta-hydroxylation, resulting in carbon-carbon cleavage and production of 2,5-dihydroxypyridine | Pseudomonas putida |