7TNF

The crystal structure of F298V CYP199A4 bound to 4-phenylbenzoic acid

Summary for 7TNF

• Entry DOI: 10.2210/pdb7tnf/pdb
• Descriptor: Cytochrome P450, PROTOPORPHYRIN IX CONTAINING FE, biphenyl-4-carboxylic acid, ... (6 entities in total)
• Functional Keywords: cytochrome p450, 4-phenylbenzoic acid, aromatic, cyp199a4 f298v mutant, oxidoreductase
• Biological source: Rhodopseudomonas palustris HaA2
• Total number of polymer chains: 1
• Total formula weight: 45413.85

Authors

Podgorski, M.,Bell, S.G. (deposition date: 2022-01-21, release date: 2023-01-04, 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 (1.539 Å)