In vitro oxidative decarboxylation of free fatty acids to terminal alkenes by two new P450 peroxygenases

Huifang Xu,Frederic Laeuffer,Cong Wang,Shengying Li,Linlin Ning,Bo Fang,Laurent Fourage,Severine Collin,Yun Wang,Jian Xu,Wenxia Yang

doi:10.1186/s13068-017-0894-x

Abstract

BackgroundP450 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.ResultsHere, we describe for the first time the expression, purification, and in vitro biochemical characterization of two new CYP152 peroxygenases, CYP-Aa162 and CYP-Sm46Δ29, 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-Sm46Δ29 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-Sm46Δ29 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 P450BSβ, emphasizing the importance of these residues in directing the decarboxylation pathway. Furthermore, the steady-state kinetic analysis of CYP-Aa162 and CYP-Sm46Δ29 revealed both cooperative and substrate inhibition behaviors which are substrate carbon chain length dependent.ConclusionsOur data identify CYP-Sm46Δ29 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.

Highlights

cytochrome P450 enzyme (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
This prompted us to re-examine the protein sequence of EKU50422 and its annotation. We found that this sequence has an extra long (~29 amino acids) N terminus with no functional annotations when compared with most other CYP152 members including those cytochrome P450 family proteins from other Staphylococcus species (Additional file 1: Figure S1)
In this study, we successfully expressed, purified, and biochemically characterized two new CYP152 peroxygenase family members, Cytochrome P450 (CYP)-Aa162 and CYP-Sm46Δ29, that are capable of decarboxylating straight-chain saturated fatty acids to yield 1-alkenes to different extents

Summary

Introduction

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. A sustainable biosynthetic route to 1-alkenes from biologically abundant feedstocks such as free fatty acids (FFAs) clearly represents a promising pathway. A fast growing number of studies have been carried out to optimize OleTJE catalysis and to understand its catalytic mechanism [16, 18,19,20,21,22,23,24,25,26,27,28]. These include the development of alternative catalytic systems (e.g., the O 2/NAD(P)H/ redox partners system [16] and the light-driven H2O2 generation system [21]) (Scheme 1), the determination of OleTJE crystal structures [18, 28], the elucidation of catalytically reactive species [19, 20], and the expansion of substrate scope to structurally different aromatic carboxylic acids [29], dionic acids (to produce dienes) [30], and even some unnatural substrates including styrene, nonane, and cyclohexane [31]

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Conclusion