The omega-hydroxylation of lauric acid: Oxidation of 12-hydroxylauric acid to dodecanedioic acid by a purified recombinant fusion protein containing P450 4A1 and NADPH-P450 reductase

The recombinant fusion protein rF450[mRat4A1/mRatOR]L1, containing the heme domain of P450 4A1 and the flavin domains of NADPH-P450 reductase, when incubated with dilaurylphosphatidylcholine (DLPC), Chaps, cytochrome b(5), and a 20-fold excess of purified NADPH-P450 reductase, catalyzes the omega-oxidation of lauric acid at a rate of about 300 nmol/min/nmol P450. This is the first report of a mammalian P450 enzyme with such a high turnover number. The resultant 12-hydroxydodecanoic acid [12-hydroxylauric acid (12-OH LA)] is further oxidized by the P450 oxygenase reaction to dodecanedioic acid (decane-1,10-dicarboxylic acid) via 12,12-dihydroxydodecanoic acid. Spectral binding studies show that 12-OH LA inhibits the binding of lauric acid to the active site of P450 with a K-i of about 1.9 mu M. The construction and expression of recombinant P450 4A1 containing a six-member polyhistidine domain at the carboxy-terminus of the protein is described, Reconstitution experiments with this purified recombinant P450 4A1, DLPC, Chaps, b(5), and purified NADPH-P450 reductase show results similar to those obtained with the purified fusion protein, albeit at lower turnover rates. The requirement for normal-phase HPLC in resolving the metabolites formed during lauric acid metabolism is demonstrated. (C) 1996 Academic Press, Inc.