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Literature summary extracted from

  • Lazar, J.T.; Shuvalova, L.; Rosas-Lemus, M.; Kiryukhina, O.; Satchell, K.J.F.; Minasov, G.
    Structural comparison of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida with PobA from other Pseudomonas spp. and other monooxygenases (2019), Acta Crystallogr. Sect. F, 75, 507-514 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
1.14.13.2 drug development p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida is a possible drug target to combat tetracycline resistance, in complex with flavin adenine dinucleotide (FAD) Pseudomonas putida

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.2 gene pobA, cloned into the ampicillin-resistant pMCSG53 vector, which contains an N-terminal polyhistidine tag followed by the Tobacco etch virus (TEV) protease cleavage site and the start codon of the pobA gene, recombinant expression in kanamycin-resistant Escherichia coli strain BL21(DE3) Pseudomonas putida

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.14.13.2 purified enzyme, sitting drop vapour diffusion method, mixing of 13.8 mg/ml protein in 100 mM Tris-HCl, pH 8.3, 5 mM 2-mercaptoethanol, and 1 mM FAD, with a reservoir solution containing 200 mM potassium thiocyanate, 100 mM bis-Tris propane, pH 6.5, and 20% w/v PEG 3350, in a 1:1 ratio, and equilibration against 0.1 ml of reservoir solution, at 19°C, X-ray diffraction structure determination and analysis at 2.2 A resolution, molecular replacement using the structure of wild-type enzyme pHBH from Pseudomonas fluorescens (PDB ID 1pbb) as a search model Pseudomonas putida
1.14.13.242 crystal structure analysis, PDB ID 5hxi, comparison to the structure of HbpA from Pseudomonas nitroreducens strain HBP1 (PDB ID 4cy8) Mesorhizobium japonicum

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas putida
-
 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 Pseudomonas putida KT-2440
-
 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
1.14.13.44 2-hydroxybiphenyl + NADH + H+ + O2 Pseudomonas nitroreducens
-
 2,3-dihydroxybiphenyl + NAD+ + H2O
-
 ?
1.14.13.242 3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2 Mesorhizobium japonicum
-
 2-(acetamidomethylidene)succinate + NAD(P)+
-
 ?
1.14.13.242 3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2 Mesorhizobium japonicum LMG 29417
-
 2-(acetamidomethylidene)succinate + NAD(P)+
-
 ?
1.14.13.242 3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2 Mesorhizobium japonicum CECT 9101
-
 2-(acetamidomethylidene)succinate + NAD(P)+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.2 Pseudomonas putida Q88H28
-
-
1.14.13.2 Pseudomonas putida KT-2440 Q88H28
-
-
1.14.13.44 Pseudomonas nitroreducens O06647
-
-
1.14.13.242 Mesorhizobium japonicum Q988D3
-
-
1.14.13.242 Mesorhizobium japonicum CECT 9101 Q988D3
-
-
1.14.13.242 Mesorhizobium japonicum LMG 29417 Q988D3
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.2 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage by TEV protease, and gel filtration, followed by dialysis and ultrafiltration Pseudomonas putida

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2
-
 Pseudomonas putida 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2
-
 Pseudomonas putida KT-2440 3,4-dihydroxybenzoate + NADP+ + H2O
-
 ?
1.14.13.2 additional information comparisons of substrate binding structure analysis mechanism Pseudomonas putida ?
-
-
1.14.13.2 additional information comparisons of substrate binding structure analysis mechanism Pseudomonas putida KT-2440 ?
-
-
1.14.13.44 2-hydroxybiphenyl + NADH + H+ + O2
-
 Pseudomonas nitroreducens 2,3-dihydroxybiphenyl + NAD+ + H2O
-
 ?
1.14.13.44 additional information comparisons of substrate binding structures, overview Pseudomonas nitroreducens ?
-
-
1.14.13.242 3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
-
 Mesorhizobium japonicum 2-(acetamidomethylidene)succinate + NAD(P)+
-
 ?
1.14.13.242 3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
-
 Mesorhizobium japonicum LMG 29417 2-(acetamidomethylidene)succinate + NAD(P)+
-
 ?
1.14.13.242 3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
-
 Mesorhizobium japonicum CECT 9101 2-(acetamidomethylidene)succinate + NAD(P)+
-
 ?
1.14.13.242 additional information comparisons of substrate binding structure analysis mechanism Mesorhizobium japonicum ?
-
-
1.14.13.242 additional information comparisons of substrate binding structure analysis mechanism Mesorhizobium japonicum LMG 29417 ?
-
-
1.14.13.242 additional information comparisons of substrate binding structure analysis mechanism Mesorhizobium japonicum CECT 9101 ?
-
-

Subunits

EC Number Subunits Comment Organism
1.14.13.2 homodimer 2 * 43000 Pseudomonas putida
1.14.13.2 More both subunits contain flavin adenine dinucleotide (FAD), which interacts with nicotinamide adenine dinucleotide phosphate (NADPH) and 4-hydroxybenzoate to form a ternary complex that allows the oxygenation of 4-hydroxybenzoate by the reduction of FAD. Each PobA chain in the structure contains 20 beta-strands and 15 alpha-helices, secondary structure analysis, overview Pseudomonas putida

Synonyms

EC Number Synonyms Comment Organism
1.14.13.2 p-hydroxybenzoate hydroxylase
-
 Pseudomonas putida
1.14.13.2 PHBH
-
 Pseudomonas putida
1.14.13.2 PobA
-
 Pseudomonas putida
1.14.13.44 2-hydroxybiphenyl 3-monooxygenase
-
 Pseudomonas nitroreducens
1.14.13.44 HbpA
-
 Pseudomonas nitroreducens
1.14.13.242 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
-
 Mesorhizobium japonicum
1.14.13.242 MHPCO
-
 Mesorhizobium japonicum
1.14.13.242 mlr6788
-
 Mesorhizobium japonicum

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.2 FAD both subunits contain flavin adenine dinucleotide (FAD), which interacts with nicotinamide adenine dinucleotide phosphate (NADPH) and 4-hydroxybenzoate to form a ternary complex that allows the oxygenation of 4-hydroxybenzoate by the reduction of FAD, binding domain structure comparisons, overview Pseudomonas putida
1.14.13.2 NADPH
-
 Pseudomonas putida
1.14.13.44 FAD binding domain structure comparisons, overview Pseudomonas nitroreducens
1.14.13.44 NADH
-
 Pseudomonas nitroreducens
1.14.13.242 FAD binding domain structure comparisons, overview Mesorhizobium japonicum
1.14.13.242 NADH
-
 Mesorhizobium japonicum

General Information

EC Number General Information Comment Organism
1.14.13.2 evolution the PobA enzyme structure is highly conserved across various organisms. Active-site residues Tyr201, Ser212, Arg214, Tyr222 and Pro293 interact with the carboxyl and phenolic components of 4-HB and are essential for its oxidative catalysis Pseudomonas putida
1.14.13.2 additional information sequence comparisons, three-dimensional enzyme structure analysis, and structure comparisons with 2-hydroxybiphenyl 3-monooxygenase (HbpA) from Pseudomonas nitroreducens and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) from Mesorhizobium japonicum, overview. Despite having only 14% similarity in their primary sequences, pairwise structure alignments of PobA from Pseudomonas putida with HbpA from Pseudomonas nitroreducens and MHPCO from Mesorhizobium japonicum reveal local similarities between these structures. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. The PobA from Pseudomonas putida is structurally very similar to PobA from Pseudomonas fluorescens and from Pseudomonas aeruginosa. Key secondary-structure elements important for catalysis, such as the betaalphabeta fold, beta-sheet wall and alpha12 helix, are conserved across this expanded class of oxygenases Pseudomonas putida
1.14.13.2 physiological function PobA is a flavin-dependent monooxygenase that utilizes one O atom from O2 to hydroxylate 4-hydroxybenzoate, while reducing the other O atom to water Pseudomonas putida
1.14.13.44 additional information sequence comparisons, three-dimensional enzyme structure analysis, and structure comparisons with 2-hydroxybiphenyl 3-monooxygenase (HbpA, PDB ID 4cy8) from Pseudomonas nitroreducens and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) from Mesorhizobium japonicum, overview. Despite having only 14% similarity in their primary sequences, pairwise structure alignments of PobA from Pseudomonas putida with HbpA from Pseudomonas nitroreducens and MHPCO from Mesorhizobium japonicum reveal local similarities between these structures. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. Key secondary-structure elements important for catalysis, such as the betaalphabeta fold, beta-sheet wall and alpha12 helix, are conserved across this expanded class of oxygenases Pseudomonas nitroreducens
1.14.13.44 physiological function HbpA hydroxylates its substrate 2-hydroxybiphenyl to 2,3-dihydroxybiphenyl Pseudomonas nitroreducens
1.14.13.242 additional information sequence comparisons, three-dimensional enzyme structure analysis, and structure comparisons with 2-hydroxybiphenyl 3-monooxygenase (HbpA) from Pseudomonas nitroreducens and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO, PDB ID 5hxi) from Mesorhizobium japonicum, overview. Despite having only 14% similarity in their primary sequences, pairwise structure alignments of PobA from Pseudomonas putida with HbpA from Pseudomonas nitroreducens and MHPCO from Mesorhizobium japonicum reveal local similarities between these structures. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. The PobA from Pseudomonas putida is structurally very similar to PobA from Pseudomonas fluorescens and from Pseudomonas aeruginosa. Key secondary-structure elements important for catalysis, such as the betaalphabeta fold, beta-sheet wall and alpha12 helix, are conserved across this expanded class of oxygenases Mesorhizobium japonicum
1.14.13.242 physiological function 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) is a flavoenzyme that catalyzes oxidative ring opening of 2-methyl-3-hydroxypyridine-5-carboxylic acid Mesorhizobium japonicum