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Literature summary for 1.14.13.163 extracted from

  • Li, H.; Xie, K.; Huang, H.; Wang, S.
    6-hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33 (2014), PLoS ONE, 9, e103324 .
    View publication on PubMedView publication on EuropePMC

Application

Application Comment Organism
synthesis development of an efficient process to transform HSP into 2,5-dihydroxypyridine (2,5-DHP) with heterologously expressed HSP hydroxylase and NADH-regenerating system, because 2,5-DHP, the product of the reaction catalyzed by HSP hydroxylase, is a valuable precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy Agrobacterium tumefaciens

Cloned(Commentary)

Cloned (Comment) Organism
DNA and amino acid sequence determination and analysis, sequence comparisons, RT-PCR expression analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Agrobacterium tumefaciens

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
90000
-
gel filtration Agrobacterium tumefaciens

Natural Substrates/ Products (Substrates)

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 Agrobacterium tumefaciens
-
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
?
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2 Agrobacterium tumefaciens S33
-
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
?

Organism

Organism UniProt Comment Textmining
Agrobacterium tumefaciens W8E0Y0 i.e. Rhizobium radiobacter
-
Agrobacterium tumefaciens S33 W8E0Y0 i.e. Rhizobium radiobacter
-

Purification (Commentary)

Purification (Comment) Organism
native enzyme 64.4fold from Agrobacterium tumefaciens strain S33 cells by ammonium sulfate fractionation, hydrophobic interaction chromatography, desalting gel filtration, and two different steps of anion exchange chromatography, followed by gel filtration. Recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, anion exchange chromatography, and ultrafiltration. During purification, all the buffers are supplemented with 0.005 mM FAD. When FAD is omitted, the purification yield is very low Agrobacterium tumefaciens

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
18.8
-
purified recombinant enzyme, pH 8.0, 30°C Agrobacterium tumefaciens
10.3
-
purified native enzyme, pH 8.0, 30°C Agrobacterium tumefaciens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information no activity with p-nitrophenol or 6-hydroxynicotinate as substrates Agrobacterium tumefaciens ?
-
?
additional information no activity with p-nitrophenol or 6-hydroxynicotinate as substrates Agrobacterium tumefaciens S33 ?
-
?
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2
-
Agrobacterium tumefaciens 2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
?
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2 oxidative decarboxylation of HSP to 2,5-dihydroxypyridine in presence of NADH and FAD Agrobacterium tumefaciens 2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
?
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2
-
Agrobacterium tumefaciens S33 2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
?
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2 oxidative decarboxylation of HSP to 2,5-dihydroxypyridine in presence of NADH and FAD Agrobacterium tumefaciens S33 2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
?

Subunits

Subunits Comment Organism
homodimer 2 * 45000, SDS-PAGE Agrobacterium tumefaciens

Synonyms

Synonyms Comment Organism
6-hydroxy-3-succinoylpyridine hydroxylase
-
Agrobacterium tumefaciens
HSP hydroxylase
-
Agrobacterium tumefaciens
NADH-dependent HSP hydroxylase
-
Agrobacterium tumefaciens
AWN88_01205 gene name, UniProt Agrobacterium tumefaciens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Agrobacterium tumefaciens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
-
Agrobacterium tumefaciens

Cofactor

Cofactor Comment Organism Structure
NADH dependent on Agrobacterium tumefaciens
FAD required for activity Agrobacterium tumefaciens

General Information

General Information Comment Organism
physiological function the key enzyme HSP hydroxylase is involved in the fused nicotine degradation pathway of the pyridine and pyrrolidine pathways Agrobacterium tumefaciens
metabolism 6-hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation. 6-Hydroxy-3-succinoylpyridine (HSP) is a key intermediate connecting the two pathways, pyridine pathway and pyrrolidine pathway, overview Agrobacterium tumefaciens