Enzyme–Substrate Complementarity Governs Access to a Cationic Reaction Manifold in the P450_BM3‐Catalysed Oxidation of Cyclopropyl Fatty Acids

Abstract

The products of cytochrome P450(BM3)-catalysed oxidation of cyclopropyl-containing dodecanoic acids are consistent with the presence of a cationic reaction intermediate, which results in efficient dehydrogenation of the rearranged probes by the enzyme. These results highlight the importance of enzyme-substrate complementarity, with a cationic intermediate occurring only when the probes used begin to diverge from ideal substrates for this enzyme. This also aids in reconciling literature reports supporting the presence of cationic intermediates with certain cytochrome P450 enzyme/substrate pairs.

Mechanistic Study of the Stereoselective Hydroxylation of [2‐2H1,3‐2H1]Butanes Catalyzed by Cytochrome P450 BM3 Variants

“…In this work, the tailored butane probes 2 and 3 used to examine the mechanism of BM3 PF and EPF variants are less sensitive than chiral ethane probes as the rotational barrier to pass the eclipsed rotamer is shown to be approximately 2.8–4.0 kcal mol −1 , that is, approximately 0–1.2 kcal mol −1 higher than that of the ethane molecule . The rotational rate constants of probes 2 or 3 are comparable to or even faster than the rebound rate of cyclopropyl fatty acid radical clock probes, estimated from the rearrangement products to be 2–3×10 10 s −1 . However, the observation of a small amount of chiral 2‐butanols with inversion of configuration generated from butane probes 2 and 3 mediated by BM3 PF and EPF variants is consistent with the results using cyclopropyl fatty acid probes mediated by cytochrome P450 BM3.…”

“…Despite the extensive applications of hydroxylation catalyzed by cytochrome P450 BM3, the mechanism of this reaction remains elusive, presumably owing to the high reactivity of Cpd I. A series of C 12 –C 16 cyclopropyl fatty acids that mimic native substrates of cytochrome P450 BM3 were developed as probes to decipher the hydroxylation mechanism mediated by this enzyme . However, less than 3–4 % of the rearranged products emerged from the rebound intermediates, indicating that the rate for the rebound of intermediate is about 2–3×10 10 s −1 , which also suggests that the oxidation mostly involves the low spin state of Cpd I with a low kinetic barrier for the C−O bond formation in the hydroxylation, or that the reactivity of the emerged Cpd I for radical rebound is too fast to be observed with the significant amount of rearranged products ,.…”

Aflatoxin and deconstruction of type I, iterative polyketide synthase function

“…The role of cations in cytochrome P450-catalyzed reactions remains controversial. 20, 21 Stereochemical and radical clock experiments can be cited in their favor as I believe this and other natural product oxidative rearrangement steps will further support.…”

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…[95][96][97] Long incubations of fatty acids with P450 BM3 can lead to the isolation of products that have undergone several rounds of oxidation, producing ketone, diol and hydroxyketone products, 89,90 but unlike systems in which sequential oxidations are required (such as P450 scc or P450 BioI , vide infra) the affinity of the enzymes for hydroxylated fatty acids is signicantly reduced over the parent fatty acids themselves. P450 BM3 is also able to epoxidise unsaturated fatty acid substrates, with the relative positioning of the alkene with regards the u-position of the fatty acid either dictating complete epoxidation, a mixture of epoxidation and hydroxylation or complete hydroxylation.…”