Cloning and functional characterization of CYP94A2, a medium chain fatty acid hydroxylase from Vicia sativa

A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydr oxylase (CYP94A2) was isolated from a Vicia sativa library. CYP94A2 display s 58% sequence identity with CYP94A1, a fatty acid omega-hydroxylase isolat ed from the same material. Heterologous expression of CYP94A2 in Saccharomy ces cerevisiae yeast strain WAT11 shows that it catalyses the hydroxylation of myristic (C14) acid with a K-m(app) of 4.0 mu M and a turnover rate num ber of 80 min(-1). In addition, lauric (C12) and palmitic (C16) acids were hydroxylated at a ten-fold lower rate, while C18 fatty acids were not oxidi zed. Remarkably, the regiospecificity of hydroxylation is different for the C12, C14, and C16 fatty acids and appears to be correlated with the length of the carbon chain. Northern blot analysis showed a low level of constitu tive expression of CYP94A2 in V. sativa seedlings. In contrast to CYP94A1, transcript accumulation of CYP94A2 was only weakly enhanced in seedlings tr eated with clofibrate or methyl jasmonate, indicating that both substrate r ange and gene regulation of the two fatty acid hydroxylases are different, (C) 1999 Academic Press.