Metabolomics for the characterization of cytochromes P450-dependent fatty acid hydroxylation reactions in Arabidopsis

Abstract

Genome sequence analysis has revealed the presence of almost infinite numbers of cytochrome P450 genes in a variety of organisms. To establish a robust experimental platform from which to explore the catalytic potential of those putative P450 proteins, we have developed a comprehensive metabolic profiling system based on Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR/MS) analysis, together with recombinant enzyme studies. Here, we report the enzymatic properties of CYP78A5, CYP78A7, CYP78A10, and CYP86C3 of Arabidopsis as short-chain fatty acid hydroxylases, with substrates including lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), and myristoleic acid (C14:1). The fatty acid hydroxylation activity of CYP78A7 was confirmed using FT-ICR/MS metabolic profiling of P450-gene-overexpressing lines of cultured Arabidopsis T87 cells. The practical application of this metabolic profiling scheme is discussed as a P450 functional characterization platform.