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; wh ich represent CYP52A4 variants naturally occurring in the yeast Candid a maltosa, were characterized with respect to their substrate specific ity. Whereas P-450Cm2 was found to catalyse lauric acid to-hydroxylati on with greater efficiency. P-450Alk3A had higher palmitic acid turnov er 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-45 0Alk3A, respectively. As shown by means of chimeric enzymes and site-d irected mutapenesis, 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 leuci ne (P-450Alk3A) led to a direct transposition of specificity suggestin g that amino acids at this site may determine the efficiency of fatty- acid hydroxylation relatively independently of other active-site resid ues. This was further supported by the finding that P-450Cm2 and P-450 Alk3A with methionine at position 527 displayed almost identical hydro xylation activities. Moreover, methionine to leucine substitutions at the corresponding alignment position in P-450Cm1 (CYP52A3), P-450Alk2A (CTP52A5) and P-450Alk5A (CYP52A9) altered the fatty-acid specificity of these enzymes, In comparison to the structure of the bacterial P-4 50BM3 (CYP102). we propose that the amino acid at position 527 may ser ve to close the substrate-binding pocket near to the harm in the fatty -acid-omega-hydroxylating P-450 of;he CYP52 family.