Enabling Aromatic Hydroxylation in a Cytochrome P450 Monooxygenase Enzyme through Protein Engineering. Issue 67 (6th October 2022)

• Record Type: Journal Article
• Authors: Coleman, Tom; Lee, Joel Z. H.; Kirk, Alicia M.; Doherty, Daniel Z.; Podgorski, Matthew N.; Pinidiya, Dilshi K.; Bruning, John B.; De Voss, James J.; Krenske, Elizabeth H.; Bell, Stephen G.
• Is Part Of: Chemistry. Volume 28:Issue 67(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-10-06
• Subjects: cytochromes -- enzyme mechanisms -- metalloenzymes -- molecular dynamics -- X-ray crystallography; Chemistry -- Periodicals; 540
• DOI: 10.1002/chem.202201895
• Languages: English
• ISSNs: 0947-6539

Abstract

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. Abstract : Structural and biochemical data demonstrate that 4‐phenylbenzoic acid binds within the active site of the cytochrome P450 monooxygenase CYP199A4, but no oxidation occurs. Protein engineering and molecular dynamic simulations establish that aromatic oxidation is enabled by a subtle shift of the substrate relative to the heme, thus improving our understanding of this important reaction.