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Literature summary extracted from

  • Chen, X.; Tang, H.; Liu, Y.; Xu, P.; Xue, Y.; Lin, K.; Cui, C.
    Purification and initial characterization of 3-hydroxybenzoate 6-hydroxylase from a halophilic Martelella strain AD-3 (2018), Front. Microbiol., 9, 1335 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.24 gene 3HB6H is encoded in a cluster that potentially encodes for gentisic acid degradation, sequence comparisons and phylogenetic analysis and tree recombinant expression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3) Martelella sp. AD-3

Protein Variants

EC Number Protein Variants Comment Organism
1.14.13.24 A308G site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme, comparison of FAD contents Martelella sp. AD-3
1.14.13.24 Q305P site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme, comparison of FAD contents Martelella sp. AD-3
1.14.13.24 Y221F site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme, comparison of FAD contents Martelella sp. AD-3
1.14.13.24 Y306H site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme, comparison of FAD contents Martelella sp. AD-3

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.14.13.24 Co2+ inhibits the enzyme activity at 0.5 mM by about 30% Martelella sp. AD-3
1.14.13.24 Cu2+ inhibits the enzyme activity at 0.5 mM by 83.2% Martelella sp. AD-3
1.14.13.24 Fe3+ inhibits the enzyme activity at 0.5 mM by about 50% Martelella sp. AD-3
1.14.13.24 Mg2+ inhibits the enzyme activity at 0.5 mM by about 50% Martelella sp. AD-3
1.14.13.24 Zn2+ inhibits the enzyme activity at 0.5 mM by 77.4% Martelella sp. AD-3

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.24 additional information
-
 additional information purified 3HB6H displays typical Michaelis-Menten kinetics, Lineweaver-Burk plots Martelella sp. AD-3
1.14.13.24 0.0337
-
 NADH pH 8.0, 30°C, mutant A308G Martelella sp. AD-3
1.14.13.24 0.038
-
 3-hydroxybenzoate pH 8.0, 30°C, mutant A308G Martelella sp. AD-3
1.14.13.24 0.0463
-
 3-hydroxybenzoate pH 8.0, 30°C, mutant Y306H Martelella sp. AD-3
1.14.13.24 0.0726
-
 3-hydroxybenzoate pH 8.0, 30°C, recombinant wild-type enzyme Martelella sp. AD-3
1.14.13.24 0.0867
-
 NADH pH 8.0, 30°C, mutant Y221F Martelella sp. AD-3
1.14.13.24 0.0913
-
 3-hydroxybenzoate pH 8.0, 30°C, mutant Q305P Martelella sp. AD-3
1.14.13.24 0.0959
-
 NADH pH 8.0, 30°C, mutant Q305P Martelella sp. AD-3
1.14.13.24 0.1037
-
 NADH pH 8.0, 30°C, mutant Y306H Martelella sp. AD-3
1.14.13.24 0.1041
-
 NADH pH 8.0, 30°C, recombinant enzyme Martelella sp. AD-3
1.14.13.24 0.1642
-
 3-hydroxybenzoate pH 8.0, 30°C, mutant Y221F Martelella sp. AD-3

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.13.24 additional information enzyme activity is highest without salinity and metal salts. Ca2+ and Mn2+ have a poor effect on enzyme activity at 0.5 mM. With salinity ranging from 0 to 85.56 mM, the increase in salinity clearly causes decreased enzyme activity, 3% activity remaining at 85.56 mM salinity Martelella sp. AD-3

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.24 3-hydroxybenzoate + NADH + H+ + O2 Martelella sp. AD-3
-
 2,5-dihydroxybenzoate + NAD+ + H2O
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.24 Martelella sp. AD-3
-
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
1.14.13.24 lipoprotein the recombinant 3HB6H expressed in Escherichia coil contains phosphatidylglycerol, while phosphatidylethanolamine is not detected Martelella sp. AD-3

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.24 recombinant N-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Martelella sp. AD-3

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.14.13.24 25.2
-
 purified recombinant enzyme, pH 8.0, 37°C Martelella sp. AD-3

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.24 3-hydroxybenzoate + NADH + H+ + O2
-
 Martelella sp. AD-3 2,5-dihydroxybenzoate + NAD+ + H2O
-
 ?

Subunits

EC Number Subunits Comment Organism
1.14.13.24 homodimer 2 * 46000, recombinant His-tagged enzyme SDS-PAGE Martelella sp. AD-3

Synonyms

EC Number Synonyms Comment Organism
1.14.13.24 3-hydroxybenzoate 6-hydroxylase
-
 Martelella sp. AD-3
1.14.13.24 3HB6H
-
 Martelella sp. AD-3

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.24 37
-
 recombinant enzyme Martelella sp. AD-3

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.14.13.24 16 50 enzyme activity is maintained at 16-37°C, and quickly lost at higher temperatures. The enzyme precipitates initially at 42°C and precipitates completely at 50°C Martelella sp. AD-3

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.13.24 1
-
 NADH pH 8.0, 30°C, mutant Y221F Martelella sp. AD-3
1.14.13.24 1.5
-
 3-hydroxybenzoate pH 8.0, 30°C, mutant Y306H Martelella sp. AD-3
1.14.13.24 1.6
-
 NADH pH 8.0, 30°C, mutant Q305P Martelella sp. AD-3
1.14.13.24 1.6
-
 3-hydroxybenzoate pH 8.0, 30°C, mutant Q305P Martelella sp. AD-3
1.14.13.24 1.6
-
 NADH pH 8.0, 30°C, mutant Y306H Martelella sp. AD-3
1.14.13.24 1.7
-
 3-hydroxybenzoate pH 8.0, 30°C, mutant Y221F Martelella sp. AD-3
1.14.13.24 2
-
 NADH pH 8.0, 30°C, mutant A308G Martelella sp. AD-3
1.14.13.24 2.3
-
 3-hydroxybenzoate pH 8.0, 30°C, mutant A308G Martelella sp. AD-3
1.14.13.24 5.7
-
 3-hydroxybenzoate pH 8.0, 30°C, recombinant wild-type enzyme Martelella sp. AD-3
1.14.13.24 7.8
-
 NADH pH 8.0, 30°C, recombinant enzyme Martelella sp. AD-3

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.24 8
-
 recombinant enzyme, phosphate buffer Martelella sp. AD-3

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.24 FAD residues 305, 306 and 308 are important for FAD binding. FAD concentration in the purified 3HB6H (0.29 mM) is 0.30 mM, and FAD/enzyme ratio is 1.02/0.05 determined by HPLC analysis Martelella sp. AD-3
1.14.13.24 NADH
-
 Martelella sp. AD-3

General Information

EC Number General Information Comment Organism
1.14.13.24 evolution sequence comparisons and phylogenetic analysis, phylogenetic tree is constructed with 3-hydroxybenzoate 6-hydroxylases, salicylate 1-hydroxylases and 3-hydroxybenzoate 4-hydroxylases from other 17 strains and confirmed that it is most closely related to 3HB6H from Pseudomonas alcaligenes strain NCIMB 9867 Martelella sp. AD-3
1.14.13.24 metabolism 3-hydroxybenzoate 6-hydroxylase can convert 3-hydroxybenzoate which is an important intermediate in the biodegradation of many aromatic hydrocarbons. 3-Hydroxybenzoate is metabolized by entering the TCA cycle through the gentisate pathway Martelella sp. AD-3
1.14.13.24 additional information residues Tyr221 and Gln305 of 3HB6H from Martelella sp. strain AD-3 are involved in substrate binding Martelella sp. AD-3
1.14.13.24 physiological function 3-hydroxybenzoate 6-hydroxylase, an NADH-dependent flavoprotein, can convert 3-hydroxybenzoate which is an important intermediate in the biodegradation of many aromatic hydrocarbons Martelella sp. AD-3

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.13.24 10.35
-
 3-hydroxybenzoate pH 8.0, 30°C, mutant Y221F Martelella sp. AD-3
1.14.13.24 11.53
-
 NADH pH 8.0, 30°C, mutant Y221F Martelella sp. AD-3
1.14.13.24 15.43
-
 NADH pH 8.0, 30°C, mutant Y306H Martelella sp. AD-3
1.14.13.24 16.68
-
 NADH pH 8.0, 30°C, mutant Q305P Martelella sp. AD-3
1.14.13.24 32.4
-
 3-hydroxybenzoate pH 8.0, 30°C, mutant Y306H Martelella sp. AD-3
1.14.13.24 59.35
-
 NADH pH 8.0, 30°C, mutant A308G Martelella sp. AD-3
1.14.13.24 60.53
-
 3-hydroxybenzoate pH 8.0, 30°C, mutant A308G Martelella sp. AD-3
1.14.13.24 62.61
-
 3-hydroxybenzoate pH 8.0, 30°C, mutant Q305P Martelella sp. AD-3
1.14.13.24 74.93
-
 NADH pH 8.0, 30°C, recombinant enzyme Martelella sp. AD-3
1.14.13.24 78.51
-
 3-hydroxybenzoate pH 8.0, 30°C, recombinant wild-type enzyme Martelella sp. AD-3