P450BM-3: Absolute configuration of the primary metabolites of palmitic acid

P450BM-3, a catalytically self-sufficient, soluble bacterial P450, contains on the same polypeptide a heme domain and a reductase domain. P450BM-3 cat alyzes the oxidation of short- and long chain, saturated and unsaturated fa tty acids. The three-dimensional structure of the heme domain both in the a bsence and in the presence of fatty acid substrates has been determined; ho wever, the fatty acid in the substrate-bound form is not adequately close t o the heme iron to permit a prediction regarding the stereoselectivity of o xidation. In the case of long chain fatty acids, the products can also serv e as substrate and be metabolized several times. In the current study, we h ave determined the absolute configuration of the three primary products of palmitic acid hydroxylation (15-, 14-, and 13-OH palmitic acid). While the 15- and 14-hydroxy compounds are produced in a highly stereoselective manne r (98% R, 2% S), the 13-hydroxy is a mixture of 72% R and 28% S, We have al so examined the binding of these three hydroxy acids to P450BM-3 and shown that only two of them (14-OH and 13-OH palmitic acid) can bind to and be fu rther metabolized by P450BM-3, The results indicate that in contrast to the flexibility of palmitoleic acid bound to the oxidized enzyme, palmitic aci d is rigidly bound in the active site during catalytic turnover. (C) 1999 A cademic Press.