Abstract
The majority of cytochrome P450 enzymes (CYPs) predominantly operate as monooxygenases, but recently a class of P450 enzymes was discovered, that can act as peroxygenases (CYP152). These enzymes convert fatty acids through oxidative decarboxylation, yielding terminal alkenes, and through α- and β-hydroxylation to yield hydroxy-fatty acids. Bioderived olefins may serve as biofuels, and hence understanding the mechanism and substrate scope of this class of enzymes is important. In this work, we report on the substrate scope and catalytic promiscuity of CYP OleTJE and two of its orthologues from the CYP152 family, utilizing α-monosubstituted branched carboxylic acids. We identify α,β-desaturation as an unexpected dominant pathway for CYP OleTJE with 2-methylbutyric acid. To rationalize product distributions arising from α/β-hydroxylation, oxidative decarboxylation, and desaturation depending on the substrate’s structure and binding pattern, a computational study was performed based on an active site complex of CYP OleTJE containing the heme cofactor in the substrate binding pocket and 2-methylbutyric acid as substrate. It is shown that substrate positioning determines the accessibility of the oxidizing species (Compound I) to the substrate and hence the regio- and chemoselectivity of the reaction. Furthermore, the results show that, for 2-methylbutyric acid, α,β-desaturation is favorable because of a rate-determining α-hydrogen atom abstraction, which cannot proceed to decarboxylation. Moreover, substrate hydroxylation is energetically impeded due to the tight shape and size of the substrate binding pocket.
Highlights
The heme-containing cytochrome P450 monooxygenases (CYPs) demonstrate broad substrate tolerance as they are vital for the metabolization of xenobiotics and drug molecules,[1] as well as for the biosynthesis of hormones.[2]
With substrate 1a, OleTJE yielded a mixture of alkene (1b) and 3-hydroxymyristic acid (1d) as sole products with an overall conversion of 11% matching previous reports.10c as the product distribution highly depended on the reaction conditions, alternative redox partners were reported to cause α-hydroxylation,11a,13,28 which was not observed here
This work provides a detailed insight into the stereoselectivity and reactivity patterns of CYP OleTJE and the CYP152 family in general
Summary
The heme-containing cytochrome P450 monooxygenases (CYPs) demonstrate broad substrate tolerance as they are vital for the metabolization of xenobiotics and drug molecules,[1] as well as for the biosynthesis of hormones.[2]. The vast majority of CYP monooxygenases require one molecule of O2, two protons, and two electrons, which are transferred one at a time from an external redox partner throughout the catalytic cycle.[1,8] A common issue of CYP monooxygenases is the uncoupling reactions, leading to the loss of redox equivalents in the form of hydrogen peroxide or superoxide anions which in turn harm the enzyme. A handful of CYPs have evolved the ability to utilize hydrogen peroxide as both electron- and oxygen-source instead, by reversing the peroxide shunt that leads to uncoupling.[9] These CYP“peroxygenases” belong to the CYP152 subfamily, do not require a redox partner, and are able to hydroxylate carboxylic acids. The Received: September 17, 2018 Revised: December 4, 2018 Published: December 6, 2018
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