Mechanistic Studies of Fatty Acid Activation by CYP152 Peroxygenases Reveal Unexpected Desaturase Activity

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.

Can a Mononuclear Iron(III)‐Superoxo Active Site Catalyze the Decarboxylation of Dodecanoic Acid in UndA to Produce Biofuels?

“…Calculations starting with hydrogen atom abstraction from the C α −H bond instead, however, were found to give substrate hydroxylation as sole products and no decarboxylation was observed. As such a mixture of products was predicted for P450 OleT JE , which was confirmed by further studies that tested a range of substrates where short‐chain fatty acids gave hydroxylation and desaturation products, whereas long‐chain unbranched fatty acids produced terminal olefins and hydroxylated fatty acids . Interestingly, the experimental work of Zhang et al .…”

“…As such am ixture of products was predicted for P450 OleT JE ,w hichw as confirmed by furthers tudies that tested ar ange of substrates where short-chain fatty acids gave hydroxylationa nd desaturation products, whereas long-chain unbranched fatty acids produced terminal olefins and hydroxylated fatty acids. [20] Interestingly,t he experimental work of Zhang et al [9] on UndA showed no evidenceo fh ydrogen atom abstraction from the C a -position of substrate when those hydrogen atoms were replaced by deuterium.…”

Controlling the Regioselectivity of Fatty Acid Hydroxylation (C10) at α‐ and β‐Position by CYP152A1 (P450Bsβ) Variants

“…CYP152A1 is a P450 peroxygenase found in Bacillus subtilis (P450Bsβ). The enzyme shows similar reactivities as OleT, but its α‐ and β‐hydroxylation activity (e. g. with preference for the β‐position on C 14 ) exceeds its activity for oxidative decarboxylation or desaturation (Scheme ) …”

“…[25] OleT (CYP152 L1) originates from Jeotgalicoccus species [3,26] and performs predominantly oxidative decarboxylation [17] besides αand β-hydroxylation or desaturation if branched carboxylic acids are used as substrates. [27] CYP152A1 is a P450 peroxygenase found in Bacillus subtilis (P450Bsβ). The enzyme shows similar reactivities as OleT, but its αand β-hydroxylation activity (e. g. with preference for the βposition on C 14 ) exceeds its activity for oxidative decarboxylation or desaturation (Scheme 1).…”

“…The enzyme shows similar reactivities as OleT, but its αand β-hydroxylation activity (e. g. with preference for the βposition on C 14 ) exceeds its activity for oxidative decarboxylation or desaturation (Scheme 1). [23,27,28] The crystal structure was solved with C 16 as substrate (PDB code: 1IZO). [30] The mixed hydroxylation-pattern, high activity and available crystal structure render CYP152A1 a suitable starting point for mutagenesis studies to head for regioselective β-hydroxylation, since no available biocatalyst allows exclusive β-hydroxylation yet.…”