The omega-hydroxlyation of lauric acid: oxidation of 12-hydroxlauric acid to dodecanedioic acid by a purified recombinant fusion protein containing P450 4A1 and NADPH-P450 reductase.
The recombinant fusion protein rF450[mRat4Al/mRatOR]L1, containing the heme domain of P450 4A1 and the flavin domains of NADPH-P450 reductase, when incubated with dilaurylphosphatidylcholine (DLPC), Chaps, cytochrome b5, 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 Ki of about 1.9 microM. 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.
Shet
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Ms
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038,USA.
Fisher
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CW
Holmans
P L
PL
Estabrook
R W
RW
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D003998:Dicarboxylic Acids / Q000378:metabolism*; D004926:Escherichia coli; D006900:Hydroxylation; D007700:Kinetics; D007850:Lauric Acids / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D003998:Dicarboxylic Acids; D007850:Lauric Acids; D011993:Recombinant Fusion Proteins; C030358:lauric acid; C031263:12-hydroxydodecanoic acid; D003577:Cytochrome P-450 Enzyme System; C036836:dodecanedioic acid; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A; D009251:NADPH-Ferrihemoprotein Reductase