Fatty acid-specific, regiospecific, and stereospecific hydroxylation by cytochrome P450 (CYP152B1) from Sphingomonas paucimobilis:: Substrate structure required for α-hydroxylation

Fatty acid alpha-hydroxylase from Sphingomonas paucimobilis is an unusual cytochrome P450 enzyme that hydroxylates the alpha-carbon of fatty acids in the presence of H2O2. Herein, we describe our investigation concerning the utilization of various substrates and the optical configuration of the alpha-hydroxyl product using a recombinant form of this enzyme. This enzyme can metabolize saturated fatty acids with carbon chain lengths of more than 10. The K-m value for pentadecanoic acid (C-15) was the smallest among the saturated fatty acids tested (C-10-C-18) and that for myristic acid (C-14) showed similar enzyme kinetics to those seen for C-15. As shorter or longer carbon chain lengths were used, K-m values increased. The turnover numbers for fatty acids with carbon chain lengths of more than 11 were of the same order of magnitude (10(3) min(-1)), but the turnover number for undecanoic acid (C-11) was less. Dicarboxylic fatty acids and methyl myristate were not metabolized, but monomethyl hexadecanedioate and omega-hydroxypalmitic acid were metabolized, though with lower turnover values. Arachidonic acid was a good substrate, comparable to C-14 or C-15. The metabolite of arachidonic acid was only alpha-hydroxyarachidonic acid. Alkanes, fatty alcohols, and fatty aldehydes were not utilized as substrates. Analysis of the optical configurations of the alpha-hydroxylated products demonstrated that the products were S-enantiomers (more than 98% enantiomerically pure). These results suggested that this P450 enzyme is strictly responsible for fatty acids and catalyzes highly stereo- and regioselective hydroxylation, where structure of omega-carbon and carboxyl carbon as well as carbon chain length of fatty acids are important for substrate-enzyme interaction.