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

Abstract

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, cytochromeb5, 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 aKiof about 1.9 μM. 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,b5, 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.