Mutagenesis and redox partners analysis of the P450 fatty acid decarboxylase OleT JE

Abstract

The cytochrome P450 enzyme OleT JE from Jeotgalicoccus sp. ATCC 8456 is capable of converting free long-chain fatty acids into α-alkenes via one-step oxidative decarboxylation in presence of H 2 O 2 as cofactor or using redox partner systems. This enzyme has attracted much attention due to its intriguing but unclear catalytic mechanism and potential application in biofuel production. Here, we investigated the functionality of a select group of residues (Arg245, Cys365, His85, and Ile170) in the active site of OleT JE through extensive mutagenesis analysis. The key roles of these residues for catalytic activity and reaction type selectivity were identified. In addition, a range of heterologous redox partners were found to be able to efficiently support the decarboxylation activity of OleT JE. The best combination turned out to be SeFdx-6 (ferredoxin) from Synechococcus elongatus PCC 7942 and CgFdR-2 (ferredoxin reductase) from Corynebacterium glutamicum ATCC 13032, which gave the highest myristic acid conversion rate of 94.4%. Moreover, Michaelis-Menton kinetic parameters of OleT JE towards myristic acid were determined.