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Literature summary extracted from

  • Yu, H.; Hausinger, R.; Tang, H.; Xu, P.
    Mechanism of the 6-hydroxy-3-succinoyl-pyridine 3-monooxygenase flavoprotein from Pseudomonas putida S16 (2014), J. Biol. Chem., 289, 29158-29170 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.163 gene hspB, phylogenetic analysis and tree, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3) Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.163 additional information
-
additional information stopped-flow spectroscopy and kinetic analysis Pseudomonas putida
1.14.13.163 0.0294
-
NADH pH 8.0, 25°C, recombinant His-tagged enzyme Pseudomonas putida
1.14.13.163 0.173
-
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate pH 8.0, 25°C, recombinant His-tagged enzyme Pseudomonas putida

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.163 4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2 Pseudomonas putida
-
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
?
1.14.13.163 4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2 Pseudomonas putida S16
-
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.163 Pseudomonas putida
-
-
-
1.14.13.163 Pseudomonas putida S16
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.163 recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Pseudomonas putida

Reaction

EC Number Reaction Comment Organism Reaction ID
1.14.13.163 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

EC Number Storage Stability Organism
1.14.13.163 -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 and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.163 4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2
-
Pseudomonas putida 2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
?
1.14.13.163 4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2
-
Pseudomonas putida S16 2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
?
1.14.13.163 additional information mass spectrometric analysis of substrates and products, with recombinant enzyme Pseudomonas putida ?
-
?
1.14.13.163 additional information mass spectrometric analysis of substrates and products, with recombinant enzyme Pseudomonas putida S16 ?
-
?

Synonyms

EC Number Synonyms Comment Organism
1.14.13.163 6-hydroxy-3-succinoyl-pyridine 3-monooxygenase
-
Pseudomonas putida
1.14.13.163 HSP 3-monooxygenase
-
Pseudomonas putida
1.14.13.163 hspB
-
Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.163 25
-
assay at Pseudomonas putida

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.13.163 5.49
-
NADH pH 8.0, 25°C, recombinant His-tagged enzyme Pseudomonas putida
1.14.13.163 7.74
-
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate pH 8.0, 25°C, recombinant His-tagged enzyme Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.163 8
-
assay at Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.163 FAD
-
Pseudomonas putida
1.14.13.163 NADH
-
Pseudomonas putida

General Information

EC Number General Information Comment Organism
1.14.13.163 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
1.14.13.163 additional information free H2O2 does not catalyze the HspB enzyme reaction Pseudomonas putida
1.14.13.163 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