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Regioselective and Stereoselective Epoxidation of n-3 and n-6 Fatty Acids by Fungal Peroxygenases.

ABSTRACT: Epoxide metabolites from n-3 and n-6 polyunsaturated fatty acids arouse interest thanks to their physiological and pharmacological activities. Their chemical synthesis has significant drawbacks, and enzymes emerge as an alternative with potentially higher selectivity and greener nature. Conversion of eleven eicosanoid, docosanoid, and other n-3/n-6 fatty acids into mono-epoxides by fungal unspecific peroxygenases (UPOs) is investigated, with emphasis on the Agrocybe aegerita (AaeUPO) and Collariella virescens (rCviUPO) enzymes. GC-MS revealed the strict regioselectivity of the n-3 and n-6 reactions with AaeUPO and rCviUPO, respectively, yielding 91%-quantitative conversion into mono-epoxides at the last double bond. Then, six of these mono-epoxides were obtained at mg-scale, purified and further structurally characterized by ¹H, ¹³C and HMBC NMR. Moreover, chiral HPLC showed that the n-3 epoxides were also formed (by AaeUPO) with total S/R enantioselectivity (ee > 99%) while the n-6 epoxides (from rCviUPO reactions) were formed in nearly racemic mixtures. The high regio- and enantioselectivity of several of these reactions unveils the synthetic utility of fungal peroxygenases in fatty acid epoxidation.

SUBMITTER: Gonzalez-Benjumea A

PROVIDER: S-EPMC8698580 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:P450 fatty acid decarboxylases represented by the unusual CYP152 peroxygenase family member OleTJE have been receiving great attention recently since these P450 enzymes are able to catalyze the simple and direct production of 1-alkenes for potential applications in biofuels and biomaterials. To gain more mechanistic insights, broader substrate spectra, and improved decarboxylative activities, it is demanded to discover and investigate more P450 fatty acid decarboxylases. RESULTS:Here, we describe for the first time the expression, purification, and in vitro biochemical characterization of two new CYP152 peroxygenases, CYP-Aa162 and CYP-Sm46?29, that are capable of decarboxylating straight-chain saturated fatty acids. Both enzymes were found to catalyze the decarboxylation and hydroxylation of a broad range of free fatty acids (C10-C20) with overlapping substrate specificity, yet distinct chemoselectivity. CYP-Sm46?29 works primarily as a fatty (lauric) acid decarboxylase (66.1 ± 3.9% 1-undecene production) while CYP-Aa162 more as a fatty (lauric) acid hydroxylase (72.2 ± 0.9% hydroxy lauric acid production). Notably, the optical spectroscopic analysis of functional CYP-Sm46?29 revealed no characteristic P450 band, suggesting a unique heme coordination environment. Active-site mutagenesis analysis showed that substitution with the proposed key decarboxylation-modulating residues, His85 and Ile170, enhanced the decarboxylation activity of CYP-Aa162 and P450BS?, emphasizing the importance of these residues in directing the decarboxylation pathway. Furthermore, the steady-state kinetic analysis of CYP-Aa162 and CYP-Sm46?29 revealed both cooperative and substrate inhibition behaviors which are substrate carbon chain length dependent. CONCLUSIONS:Our data identify CYP-Sm46?29 as an efficient OleTJE-like fatty acid decarboxylase. Oxidative decarboxylation chemoselectivity of the CYP152 decarboxylases is largely dependent upon the carbon chain length of fatty acid substrates and their precise positioning in the enzyme active site. Finally, the kinetic mode analysis of the enzymes could provide important guidance for future process design.

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Regioselective and Stereoselective Epoxidation of n-3 and n-6 Fatty Acids by Fungal Peroxygenases.

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