DIFFERENT MECHANISMS OF REGIOSELECTION OF FATTY-ACID HYDROXYLATION BYLAURATE (OMEGA-1)-HYDROXYLATING P450S, P450 2C2 AND P450 2E1

P450 2C2 as well as P450 2E1 [Fukuda, T. et at. (1993) J. Biochem. 113 , 7-12] catalyzed the hydroxylation of medium chain fatty acids, altho ugh the regioselectivity of substrates of the former contrasted with t hat of the latter. Whereas P450 2E1 hydroxylated C-9-C-18 fatty acids at the omega-1 position and to a much lesser extent at the omega and o mega-2 positions, P450 2C2 hydroxylated C-9-C-13 fatty acids at differ ent positions dependent on the chain length of fatty acids. Among the fatty acids used as the substrate, undecanoate was hydroxylated at the omega-1 position almost exclusively by P450 2C2. The proportion of om ega-hydroxylated products produced by P450 2C2 was markedly increased with decreasing chain length of fatty acids, while the hydroxylation p ositions were enlarged to the omega-3 position with tridecanoate. When the conserved Thr at the putative distal helix was replaced with Ser, the substrate regioselectivity of the two P450s was affected in diffe rent manners. The mutation of P450 2C2 did not change the hydroxylatio n positions of C-9-C-12 fatty acids, but caused a significant decrease in the proportion of the omega-1 hydroxy analog in the total products . In sharp contrast to P450 2C2, the mutated P450 2E1 gave additional products to those with the wild-type P450, and the number of different products increased with increasing chain length of the fatty acids. T hus, the products of palmitate hydroxylation were identified as omega- 1, omega-2, omega-3, omega-4, omega-5, omega-6, and omega-7 monohydrox y isomers using gas chromatogr aphy-electron impact mass spectrometry. From these findings, (i) P450 2C2 shows the substrate selectivity of undecanoate 10-hydroxylation, whereas P450 2E1 has the activity of fat ty acid omega-1 hydroxylation, and (ii) P450 2E1 is speculated to have a larger substrate pocket near the distal heme surface than P450 2C2 and the gamma-methyl Group of the conserved Thr may contribute to the limitation of the hydroxylation position in different ways in the two P450s.