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 omega-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-omega-hydroxylating P-450 of the CYP52 family.
Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany. tzim@mti-n.mti.uni-jena.de
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D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002175:Candida / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D005227:Fatty Acids / Q000378:metabolism*; D006900:Hydroxylation; D007850:Lauric Acids / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D019308:Palmitic Acid / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity
D005227:Fatty Acids; D007850:Lauric Acids; D011993:Recombinant Fusion Proteins; C030358:lauric acid; D019308:Palmitic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases