Mutual conversion of fatty‐acid substrate specificity by a single amino‐acid exchange at position 527 in P‐450Cm2 and P‐450Alk3A

The two eukaryotic fatty‐acid hydroxylases P‐450Cm2 and P‐450Alk3A, which represent CYP52A4 variants naturally occurring in the yeast Candida maltosa, were characterized with respect to their substrate specificity. Whereas P‐450Cm2 was found to catalyse lauric acid ω‐hydroxylation with greater efficiency, P‐450Alk3A had higher palmitic acid turnover numbers compared to P‐450Cm2, resulting in ratios of lauric acid to palmitic acid turnover rates of nearly 11 and 3 for P‐450Cm2 and P‐450Alk3A, respectively. As shown by means of chimeric enzymes and site‐directed mutagenesis, the key residue determining these differences in substrate specificity was found to be a single amino acid at position 527. Interestingly, the mutual exchange of valine (P‐450Cm2) and leucine (P‐450Alk3A) led to a direct transposition of specificity, suggesting that amino acids at this site may determine the efficiency of fatty‐acid hydroxylation relatively independently of other active‐site residues. This was further supported by the finding that P‐450Cm2 and P‐450Alk3A with methionine at position 527 displayed almost identical hydroxylation activities. Moreover, methionine to leucine substitutions at the corresponding alignment position in P‐450Cm1 (CYP52A3), P‐450Alk2A (CYP52A5) and P‐450Alk5A (CYP52A9) altered the fatty‐acid specificity of these enzymes. In comparison to the structure of the bacterial P‐450BM3 (CYP102), we propose that the amino acid at position 527 may serve to close the substrate‐binding pocket near to the haem in the fatty‐acid‐ω‐hydroxylating P‐450 of the CYP52 family.