Literature summary for 1.14.13.2 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 .

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Application

Application	Comment	Organism
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)

Cloned (Comment)	Organism
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)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
4-hydroxybenzoate + NADPH + H+ + O2	Pseudomonas putida	-	3,4-dihydroxybenzoate + NADP+ + H2O	-	?
4-hydroxybenzoate + NADPH + H+ + O2	Pseudomonas putida KT-2440	-	3,4-dihydroxybenzoate + NADP+ + H2O	-	?

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas putida	Q88H28	-	-
Pseudomonas putida KT-2440	Q88H28	-	-

Purification (Commentary)

Purification (Comment)	Organism
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
4-hydroxybenzoate + NADPH + H+ + O2	-	Pseudomonas putida	3,4-dihydroxybenzoate + NADP+ + H2O	-	?
4-hydroxybenzoate + NADPH + H+ + O2	-	Pseudomonas putida KT-2440	3,4-dihydroxybenzoate + NADP+ + H2O	-	?
additional information	comparisons of substrate binding structure analysis mechanism	Pseudomonas putida	?	-	-
additional information	comparisons of substrate binding structure analysis mechanism	Pseudomonas putida KT-2440	?	-	-

Subunits

Subunits	Comment	Organism
homodimer	2 * 43000	Pseudomonas putida
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

Synonyms	Comment	Organism
p-hydroxybenzoate hydroxylase	-	Pseudomonas putida
PHBH	-	Pseudomonas putida
PobA	-	Pseudomonas putida

Cofactor

Cofactor	Comment	Organism	Structure
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
NADPH	-	Pseudomonas putida

General Information

General Information	Comment	Organism
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
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
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