7JXB

The crystal structure of 4-(3'-methoxyphenyl)benzoic acid-bound CYP199A4

Summary for 7JXB

• Entry DOI: 10.2210/pdb7jxb/pdb
• Descriptor: Cytochrome P450, 3'-methoxy[1,1'-biphenyl]-4-carboxylic acid, PROTOPORPHYRIN IX CONTAINING FE, ... (5 entities in total)
• Functional Keywords: cytochrome, p450, monooxygenase, heme, oxygen insertion, oxidoreductase
• Biological source: Rhodopseudomonas palustris (strain HaA2)
• Total number of polymer chains: 1
• Total formula weight: 45467.61

Authors

Doherty, D.Z.,Bell, S.G.,Bruning, J. (deposition date: 2020-08-27, release date: 2022-03-09, Last modification date: 2023-10-25)

Primary citation

Coleman, T.,Lee, J.Z.H.,Kirk, A.M.,Doherty, D.Z.,Podgorski, M.N.,Pinidiya, D.K.,Bruning, J.B.,De Voss, J.J.,Krenske, E.H.,Bell, S.G.
Enabling Aromatic Hydroxylation in a Cytochrome P450 Monooxygenase Enzyme through Protein Engineering.
Chemistry, 28:e202201895-e202201895, 2022

PubMed Abstract: The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C-H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4-cyclohexylbenzoic acid but not the aromatic oxidation of 4-phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4-benzyl-, 4-phenoxy- and 4-benzoyl-benzoic acid and methoxy-substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4-benzylbenzoic acid. The methoxy-substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4-(3'-methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4-phenylbenzoic acid. 4-Phenoxy- and 4-benzoyl-benzoic acid bound with the ether or ketone oxygen atom hydrogen-bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4-phenylbenzoic acid; however the F182L mutant was able to catalyse 4-phenylbenzoic acid oxidation generating 2'-hydroxy-, 3'-hydroxy- and 4'-hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild-type enzyme, the phenyl ring of 4-phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur.

PubMed: 36043399
DOI: 10.1002/chem.202201895

Experimental method

X-RAY DIFFRACTION (1.655 Å)