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Literature summary for 1.14.99.15 extracted from

  • Bell, S.; Yang, W.; Tan, A.; Zhou, R.; Johnson, E.; Zhang, A.; Zhou, W.; Rao, Z.; Wong, L.
    The crystal structures of 4-methoxybenzoate bound CYP199A2 and CYP199A4: Structural changes on substrate binding and the identification of an anion binding site (2012), Dalton Trans., 41, 8703-8714.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli strain BL21(DE3) Rhodopseudomonas palustris
wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Rhodopseudomonas palustris

Crystallization (Commentary)

Crystallization (Comment) Organism
enzyme CYP199A2 bound to substrate 4-methoxybenzoate, X-ray diffraction structure determination and analysis at 1.8 A resolution Rhodopseudomonas palustris
enzyme CYP199A4 free and bound to substrate 4-methoxybenzoate, hanging drop vapour diffusion method, for the free enzyme: mixing of 0.001 ml of protein solution containing 50 mg/ml protein in 20 mM HEPES, pH 7.4, 150 mM KCl, 1 mM DTT, with 0.001 ml of reservoir solution containing 0.1 M Bis-Tris, pH 5.5, 1.45-1.5 M ammonium sulfate, and 0.1 M sodium chloride, and equilibration against 0.2 ml of reservoir solution, 20°C, 1 week, for the substrate-bound enzyme: mixing of 0.001 ml of protein solution containing 40 mg/ml protein in 20 mM HEPES, pH 7.4, 150 mM KCl, and 10 mM 2-mercaptoethanol and saturated with 4-methoxybenzoate, with 0.001 ml of reservoir solution containing 0.1 M Bis-Tris, pH 5.5, 1.45 M ammonium sulfate, and 0.1 M sodium chloride, and equilibration against 0.2 ml of reservoir solution, 20°C, 2 weeks, X-ray diffraction structure determination and analysis at 2.6 A and 2.0 A resolution, respectively Rhodopseudomonas palustris

Protein Variants

Protein Variants Comment Organism
F185I site-directed mutagenesis, the mutation reduces the spin state shift from low- to high-spin on the addition of 4-methoxybenzoate by about 25% compared to the wild-type enzyme, the mutant shows reduced NADH consumption and product formation Rhodopseudomonas palustris
F185V site-directed mutagenesis, the mutation reduces the spin state shift from low- to high-spin on the addition of 4-methoxybenzoate by about 35% compared to the wild-type enzyme, the mutant shows reduced NADH consumption and product formation Rhodopseudomonas palustris
R243T site-directed mutagenesis, the mutation reduces the spin state shift from low- to high-spin on the addition of 4-methoxybenzoate by about 25% compared to the wild-type enzyme, the mutant shows reduced NADH consumption and product formation Rhodopseudomonas palustris
R92E site-directed mutagenesis, the spin state shift is similar to the wild-type enzyme, but the mutant shows 3fold higher KD for the substrate, NADH consumption is reduced 9fold compared to the wild-type enzyme Rhodopseudomonas palustris
S95V site-directed mutagenesis, the mutation abolishes the spin state shift from low- to high-spin on the addition of 4-methoxybenzoate and results in a 99% drop in the NADH consumption rate comared to the wild-type enzyme Rhodopseudomonas palustris

Inhibitors

Inhibitors Comment Organism Structure
Cl- addition of chloride to the phosphate buffered samples weakens substrate binding, chloride binding site structure, overview; addition of chloride to the phosphate buffered samples weakens substrate binding, chloride binding site structure, overview Rhodopseudomonas palustris

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ heme enzyme Rhodopseudomonas palustris

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4-methoxybenzoate + AH2 + O2 Rhodopseudomonas palustris
-
4-hydroxybenzoate + formaldehyde + A + H2O
-
?
4-methoxybenzoate + AH2 + O2 Rhodopseudomonas palustris HaA2
-
4-hydroxybenzoate + formaldehyde + A + H2O
-
?
4-methoxybenzoate + AH2 + O2 Rhodopseudomonas palustris CGA009
-
4-hydroxybenzoate + formaldehyde + A + H2O
-
?

Organism

Organism UniProt Comment Textmining
Rhodopseudomonas palustris Q2IU02
-
-
Rhodopseudomonas palustris Q6N8N2
-
-
Rhodopseudomonas palustris CGA009 Q6N8N2
-
-
Rhodopseudomonas palustris HaA2 Q2IU02
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant enzyme from Escherichia coli strain BL21(DE3) by two different steps of anion exchange chromatography, followed by gel filtration Rhodopseudomonas palustris
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by two different steps of anion exchange chromatography, followed by gel filtration Rhodopseudomonas palustris

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-ethylbenzoate + AH2 + O2 computational docking study Rhodopseudomonas palustris ?
-
?
4-ethylbenzoate + AH2 + O2 computational docking study Rhodopseudomonas palustris CGA009 ?
-
?
4-methoxybenzoate + AH2 + O2
-
Rhodopseudomonas palustris 4-hydroxybenzoate + formaldehyde + A + H2O
-
?
4-methoxybenzoate + AH2 + O2
-
Rhodopseudomonas palustris HaA2 4-hydroxybenzoate + formaldehyde + A + H2O
-
?
4-methoxybenzoate + AH2 + O2
-
Rhodopseudomonas palustris CGA009 4-hydroxybenzoate + formaldehyde + A + H2O
-
?
4-methoxybenzoate + reduced palustrisredoxin + O2
-
Rhodopseudomonas palustris 4-hydroxybenzoate + formaldehyde + oxidized palustrisredoxin + H2O
-
?
4-methoxybenzoate + reduced palustrisredoxin + O2
-
Rhodopseudomonas palustris CGA009 4-hydroxybenzoate + formaldehyde + oxidized palustrisredoxin + H2O
-
?
additional information CYP199A2 oxidizes para-substituted benzoic acids with almost total NADH-to-product conversion with the highest activity being observed in the oxidative demethylation of 4-methoxybenzoate. Exclusive attack by CYP199A2 and CYP199A4 at the methoxy methyl group, leading to demethylation to form 4-hydroxybenzoate as the only product Rhodopseudomonas palustris ?
-
?
additional information exclusive attack by CYP199A2 and CYP199A4 at the methoxy methyl group, leading to demethylation to form 4-hydroxybenzoate as the only product Rhodopseudomonas palustris ?
-
?
additional information exclusive attack by CYP199A2 and CYP199A4 at the methoxy methyl group, leading to demethylation to form 4-hydroxybenzoate as the only product Rhodopseudomonas palustris HaA2 ?
-
?
additional information CYP199A2 oxidizes para-substituted benzoic acids with almost total NADH-to-product conversion with the highest activity being observed in the oxidative demethylation of 4-methoxybenzoate. Exclusive attack by CYP199A2 and CYP199A4 at the methoxy methyl group, leading to demethylation to form 4-hydroxybenzoate as the only product Rhodopseudomonas palustris CGA009 ?
-
?
veratrate + AH2 + O2
-
Rhodopseudomonas palustris ?
-
?
veratrate + AH2 + O2
-
Rhodopseudomonas palustris HaA2 ?
-
?

Synonyms

Synonyms Comment Organism
CYP199A2
-
Rhodopseudomonas palustris
CYP199A4
-
Rhodopseudomonas palustris
RPA1871
-
Rhodopseudomonas palustris

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Rhodopseudomonas palustris

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
37.9
-
4-Methoxybenzoate pH 7.4, 30°C Rhodopseudomonas palustris

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4
-
assay at Rhodopseudomonas palustris

Cofactor

Cofactor Comment Organism Structure
heme
-
Rhodopseudomonas palustris
additional information the CYP enzyme surface closest to the heme (proximal surface) is the probable ferredoxin binding site Rhodopseudomonas palustris
palustrisredoxin i.e. Pux, RPA1872, with [2Fe-2S] cluster Rhodopseudomonas palustris

General Information

General Information Comment Organism
evolution the enzyme belongs to the superfamily of heme-dependent cytochrome P450 monooxygenases Rhodopseudomonas palustris
additional information substrate-enzyme structure, the enzyme possesses a clearly defined substrate access channel that is formed between the BC loop and the G and G' helices, overview. The 4-methoxybenzoate-bound enzyme has a closed conformation, in contrast to the substrate-free form of CYP199A2 where an obvious substrate access channel is observed. The switch from an open to a closed conformation arises from pronounced residue side-chain movements and alterations of ion pair and hydrogen bonding interactions at the entrance of the access channel. A chloride ion bound just inside the protein surface caps the entrance to the active site and protects the substrate and the heme from the external solvent. The substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of the enzyme for oxidative demethylation of 4-methoxybenzoate Rhodopseudomonas palustris
additional information the 4-methoxybenzoate-bound enzyme has a closed conformation. The substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of the enzyme for oxidative demethylation of 4-methoxybenzoate, involvement of hydrophobic (Phe185) and hydrophilic (Arg92, Ser95 and Arg243) amino acid residues in the binding of para-substituted benzoates Rhodopseudomonas palustris
physiological function CYP199A2 is involved in the degradation of ligninolic compounds by the organism Rhodopseudomonas palustris