Mechanism and Catalytic Potential of a Cytochrome P450 (OleT) that Catalyzes C‐C Bond Scission

Abstract

OleT (CYP152L1) is a recently discovered cytochrome P450 that is structurally similar to peroxide dependent P450s that catalyze fatty acid hydroxylations. OleT has been shown to catalyze the cleavage of n chain length fatty acids to form n‐1 alkenes through C‐C bond scission of the terminal carboxylate. Using headspace GC‐MS and FTIR with isotopically labeled substrates, we have confirmed that OleT metabolizes eicosanoic acid to 1‐nonadecene, forming carbon dioxide as a co‐product of the reaction. No oxygen deriving from peroxide is observed in either product. In order to probe the mechanistic deviation of OleT from P450 peroxygenases, we have employed a “decoy” approach where a short chain length fatty acid provides the acidic group necessary for peroxide heterolysis, and also allows access of small molecule probe substrates. Using this approach, we confirm that OleT also catalyzes epoxidation and hydroxylation reactions. Products derived from the metabolism of radical clock substrates directly show that OleT is capable of efficient oxygen rebound with radical lifetimes that are similar to other P450 monooxygenases. A site‐directed mutant, in which the acidic group is instead provided by the protein framework, circumvents the need for a substrate carboxylate, and shows efficient metabolism of a wide range of hydrocarbon substrates.Support or Funding InformationThis work is supported by startup funds from the University of South Carolina, and funding from the U.S.C. Office of Research through the Aspire program to T.M.M.