Role of Leu188 in the Fatty Acid Hydroxylase Activity of CYP102A1 from Bacillus megaterium

• The role of Leu188 residue for fatty acid hydroxylation by CYP102A1 was examined.
• L188Q and L188 P mutants increased hydroxylase activity toward C10-C13 fatty acids.
• L188 is important for the hydroxylation activity toward fatty acids with C10-C13.
• 19 mutants of Leu188 showed similar overall hydroxylation rates for myristic acid.
• L188 residue is important for the regioselectivity of myristic acid.

P450 BM3 (CYP102A1) from Bacillus megaterium catalyzes the subterminal hydroxylation of fatty acids with 12-22 carbons at the ω-1, ω-2 and ω-3 positions. Several amino acids located at the substrate channel and active sites are known to be important for the catalytic activity of CYP102A1. The L188 residue at the C-terminus of α-helix F undergoes a large shift upon substrate binding and has frequently been found in different combinations of multiple mutations showing enhanced and altered activities. In this study, we examined the role of the L188 residue by comparing the catalytic activities of wild-type CYP102A1 and 19 mutants of L188. The mutants were made with site-directed mutagenesis and functionally expressed in Escherichia coli. The enzymatic properties of the mutants for a set of fatty acids (C10-C16) were compared to the properties of the wild-type. L188Q and L188 P mutants showed especially strong increases in hydroxylase activity toward C10-C13 fatty acids, although they did not have activity changes for C14-C16 fatty acids. Although most mutants showed very similar overall hydroxylation rates for myristic acid, 14 mutants showed apparent changes in the regioselectivity of hydroxylation with a preference for the ω-3 position over the ω-1 position. A possible role for the L188 residue has been discussed in the context of the structure and function of CYP102A1.

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