The cytochrome P450 enzyme, CYP199A4 from Rhodopseudomonas palustris strain HaA2, is able to oxidize parasubstituted benzoic acids. This enzyme was used to compare aromatic versus aliphatic C−H bond oxidation, common reactions catalyzed by the P450 superfamily of heme monooxygenases. CYP199A4 was able to bind 4-phenylbenzoic acid and 4cyclohexylbenzoic acid, and the crystal structures demonstrated that both substrates are bound within the active site in a similar fashion. Despite this, while 4-cyclohexylbenzoic acid was efficiently hydroxylated, no detectable enzyme catalyzed oxidation of the aromatic 4-phenylbenzoic acid was observed. The selectivity of 4-cyclohexylbenzoic acid oxidation favored C−H bond abstraction at one of the β-sites in an enantioselective fashion (66%, 95:5 er), over C−H bond abstraction at the benzylic position (33%). In addition, unlike the oxidation of smaller alkyl-substituted benzoic acids (4-ethyl-and 4-isopropyl-), little or no desaturation of the cyclohexyl ring to give an alkene was detected (∼1%). Molecular dynamics simulations suggested that the cyclohexyl ring of 4cyclohexylbenzoic acid was able to achieve a suitable orientation to enable efficient C−H bond abstraction and oxidation by the enzyme at the expected positions. In contrast, when the distance and angle of attack were considered, the alignment of the phenyl ring of 4-phenylbenzoic acid rarely attained a productive geometry for aromatic oxidation to occur. Overall, these results illustrate the chemoselectivity that may arise due to the different geometrical requirements for efficient aromatic oxidation versus aliphatic C− H bond hydroxylation by cytochrome P450 enzymes.

The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon‐hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4‐ethyl‐ and 4‐isopropyl‐ benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4‐cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4‐n‐propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cβ carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4‐n‐butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4‐isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cβ position (41%). The oxidation of 4‐n‐propylbenzoic acid by the F298 V mutant of CYP199A4 occurred with no hydroxylation at Cβ and a significant increase in metabolites arising from desaturation (73%). The X‐ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4‐n‐propylbenzoic acid in the F298 V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme.

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.

The steroid binding CYP142 cytochrome P450 enzymes of Mycobacterium species are involved in the metabolism of cholesterol and its derivatives. The equivalent enzyme from Mycobacterium ulcerans was studied to compare the degree of functional conservation between members of this CYP family. We compared substrate binding of the CYP142A3 enzymes of M. ulcerans and M. marinum and CYP142A1 from M. tuberculosis using UV–vis spectroscopy. The catalytic oxidation of cholesterol derivatives by all three enzymes was undertaken. Both CYP142A3 enzymes were structurally characterized by X-ray crystallography. The amino acid sequences of the CYP142A3 enzymes are more similar to CYP142A1 from M. tuberculosis than CYP142A2 from Mycolicibacterium smegmatis. Both CYP142A3 enzymes have substrate binding properties, which are more resemblant to CYP142A1 than CYP142A2. The cholest-4-en-3-one-bound X-ray crystal structure of both CYP142A3 enzymes were determined at a resolution of <1.8 Å, revealing the substrate binding mode at a high level of detail. The structures of the cholest-4-en-3-one binding CYP142 enzymes from M. ulcerans and M. marinum demonstrate how the steroid binds in the active site of these enzymes. They provide an explanation for the high selectivity of the enzyme for terminal methyl C–H bond oxidation to form 26-hydroxy derivatives. These enzymes in pathogenic Mycobacterium species are candidates for inhibition. The work here demonstrates that similar drug molecules could target these CYP142 enzymes from different species in order to combat Buruli ulcer or tuberculosis.

Cholesterol catabolism is an important survival mechanism for the pathogenic Mycobacterium tuberculosis. Various other mycobacteria degrade not only cholesterol but plant sterols such as sitosterol and campesterol. In this work...