Exploring the Factors which Result in Cytochrome P450 Catalyzed Desaturation Versus Hydroxylation

Abstract

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.

Engineering C–C Bond Cleavage Activity into a P450 Monooxygenase Enzyme

“…The position of the benzoic acid moieties of both were essentially the same as those of other substrate-bound X-ray crystal CYP199A4 structures (Figure ). ,,− The electron density for the ligand in the active site of the structure with JCM2 did not match that of the added substrate. It was modeled as terephthalic acid with additional water molecules within the active site (Figure S24 and Table S2).…”

“…4-Acetylbenzoic acid bound to CYP199A4 with significantly lower shift to the high-spin ferric state and binding affinity ( K d of 140 ± 5 μM) than 4- n -propylbenzoic acid ( ≥ 95% high-spin, 0.54 μM) and other comparable alkyl-substituted benzoic acids (Table ). , …”

“…Rates are expressed as mol (mol CYP) −1 min –1 which is abbreviated as min –1 . 4- n -Propylbenzoic acid with WT CYP199A4 data is shown for comparison …”

“…We hypothesized that mutation of the CYP199A4 enzyme may enable the JCM1 and JCM2 substrates to alter their position within the active site of the enzyme to better enable catalytic C–C bond cleavage. The F182L and F298V mutations were chosen as, in previous work, they have demonstrated alterations in the active-site orientation of other substrates, as confirmed through X-ray structures and MD simulations. , These changes enabled the oxidation of substrates which were not oxidized by the WT enzyme or resulted in an altered product distribution.…”

“…These structures have been used to elucidate binding modes and to rationalize the observed activities. − , The CYP199A4 enzyme oxidizes its substrates almost exclusively at the substituent para to the carboxylate group and hence is an ideal model system to investigate different aspects of the mechanisms of P450 reactions . For example, we have used the CYP199A4 enzyme to investigate catalytic epoxidation, sulfoxidation, and aromatic oxidation by CYP enzymes. − In addition, it can hydroxylate and desaturate the alkyl substituents of substrates such as 4- n -propylbenzoic acid …”