A conservative amino acid substitution alters the regiospecificity of CYP94A2, a fatty acid hydroxylase from the plant Vicia sativa

Fatty acid omega -hydroxylation is involved in the biosynthesis of the plan t cuticle, formation of plant defense signaling molecules, and possibly in the rapid catabolism of free fatty acids liberated under stress conditions. CYP94A2 is a cytochrome P450-dependent medium-chain fatty acid hydroxylase that was recently isolated from Vicia sativa, Contrary to CYP94A1 and CYP8 6A1, two other fatty acid hydroxylases previously characterized in V. sativ a and Arabidopsis thaliana, CYP94A2 is not a strict omega -hydroxylase, but exhibits chain-length-dependent regioselectivity of oxidative attack. Sequ ence alignments of CYP94A2 with CYP94A1 and molecular modeling studies sugg ested that F494, located in SRS-B (substrate recognition site) was involved in substrate recognition and positioning. Indeed, a conservative amino aci d substitution at that position markedly altered the regiospecificity of CY P94A2. The observed shift from omega toward omega -1 hydroxylation was prom inent with lauric acid as substrate and declined with increasing fatty acid chain length. (C) 2001 Academic Press.