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

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Item Type:	Article
Title:	Mutual conversion of fatty-acid substrate specificity by a single amino-acid exchange at position 527 in P-450Cm2 and P-450Alk3A
Creators Name:	Zimmer, T., Scheller, U., Takagi, M. and Schunck, W.H.
Abstract:	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.
Keywords:	Cytochrome P-450, Active Site, Fatty-Acid Substrate Specificity, Site-Directed Mutagenesis
Source:	European Journal of Biochemistry
ISSN:	0014-2956
Publisher:	Blackwell Publishing
Volume:	256
Number:	2
Page Range:	398-403
Date:	1 September 1998
Official Publication:	https://doi.org/10.1046/j.1432-1327.1998.2560398.x
PubMed:	View item in PubMed

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