Abstract
BackgroundThe CYP152 family member OleTJE from Jeotgalicoccus sp. ATCC 8456 has been well-known to catalyze the unusual one-step decarboxylation of free fatty acids towards the formation of terminal alkenes. Efforts to tune up its decarboxylation activity for better production of biological alkenes have been extensively explored via approaches such as site-directed mutagenesis and electron source engineering, but with limited success. To gain more insights into the decarboxylation mechanism and reaction bifurcation (decarboxylation versus hydroxylation), we turned to an alternative approach to explore the natural CYP152 resources for a better variety of enzyme candidates.ResultsWe biochemically characterized three new P450 fatty acid decarboxylases including OleTJH, OleTSQ and OleTSA, with respect to their substrate specificity, steady-state kinetics, and salt effects. These enzymes all act as an OleTJE-like fatty acid decarboxylase being able to decarboxylate a range of straight-chain saturated fatty acids (C8–C20) to various degrees. Site-directed mutagenesis analysis to the lower activity P450 enzyme OleTSA revealed a number of key amino acid residues within the substrate-binding pocket (T47F, I177L, V319A and L405I) that are important for delicate substrate positioning of different chain-length fatty acids and thus the decarboxylation versus hydroxylation chemoselectivity, in particular for the mid-chain fatty acids (C8–C12). In addition, the three new decarboxylases exhibited optimal catalytic activity and stability at a salt concentration of 0.5 M, and were thus classified as moderate halophilic enzymes.ConclusionThe P450 fatty acid decarboxylases OleTJE, OleTJH, OleTSQ and OleTSA belong to a novel group of moderate halophilic P450 enzymes. OleTJH from Jeotgalicoccus halophilus shows the decarboxylation activity, kinetic parameters, as well as salt tolerance and stability that are comparable to OleTJE. Site-directed mutagenesis of several key amino acid residues near substrate-binding pocket provides important guidance for further engineering of these P450 fatty acid decarboxylases that hold promising application potential for production of α-olefin biohydrocarbons.
Highlights
The CYP152 family member OleTJE from Jeotgalicoccus sp
To explore more cytochrome P450 enzyme (P450) fatty acid decarboxylases (FADCs) that may possess greater decarboxylation activity and/or selectivity, we built a phylogenetic tree (Fig. 2) based on the protein sequences of the two known P450 FADCs OleTJE (CYP152L1) and CYPSm46Δ29 (CYP152L2), and their homologous sequences with a sequence identity higher than 60%
These sequences mostly originate from the genera of Jeotgalicoccus, Staphylococcus, and Salinicoccus, which are well-known microorganisms associated with halophilicity or salt tolerance [35,36,37,38]
Summary
The CYP152 family member OleTJE from Jeotgalicoccus sp. ATCC 8456 has been well-known to catalyze the unusual one-step decarboxylation of free fatty acids towards the formation of terminal alkenes. Efforts to tune up its decarboxylation activity for better production of biological alkenes have been extensively explored via approaches such as site-directed mutagenesis and electron source engineering, but with limited success. The biofuel-related P450 fatty acid decarboxylases (FADCs), such as OleTJE from Jeotgalicoccus sp. ATCC 8456 [11] and CYP-Sm46 from Staphylococcus massiliensis S46 [12], belong to the CYP152 peroxygenase family This family of P450 enzymes utilizes H2O2, instead of O2 which is employed by most of P450 monooxygenases, as the oxidant to support the unique oxidative decarboxylation reactions (Fig. 1) that convert the Cn (n = 4–22) chain length free fatty acids (FFAs) into Cn−1 chain length 1-alkenes (i.e., α-olefins) [13, 14]. Since α-olefins are both excellent biofuel molecules and useful precursors of lubricants, detergents and other chemicals [15, 16], P450 FADCs hold promising application potential for production of biological α-olefins
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