Replacement of Thr-303 of P450 2E1 with serine modifies the regioselectivity of its fatty acid hydroxylase activity.
Threonine-303 of rabbit P450 2E1, which is putatively located at the distal heme surface, was replaced by serine and valine via site-directed mutagenesis. In the oxidized state, the Ser-mutated P450 exhibited a low- and high-spin mixed-type (low > high) absorption spectrum, whereas the Val-mutated P450, like the wild-type P450, exhibited a nearly high-spin type spectrum. The reduced CO complexes of the Ser- and Val-mutated P450s, as well as that of the wild-type P450, showed a Soret absorption maximum at 452 nm. Both mutated P450s were active in the hydroxylation of C10 to C18 fatty acids at somewhat lower rates than the wild-type P450. The Val-mutated P450 gave the same two products (the major one is probably the omega-1 hydroxy analog) as the wild-type P450, while additional products were formed on incubation with C11 to C17 fatty acids as substrates of the Ser-mutated P450; a total of four products was detected for each of the C12 to C15 fatty acids, and three for each of the C11, C16, and C17 homologues. The metabolites of laurate were determined by GC-MS analysis to be the omega-1, omega-2, omega-3, and omega-4 hydroxy counterparts. The Ser-mutated P450 hydroxylated drug substrates at almost the same rates as the wild-type P450, while the mutation to valine significantly lowered the drug hydroxylase activities.(ABSTRACT TRUNCATED AT 250 WORDS)
Fukuda
T
T
Department of Veterinary Science, University of Osaka Prefecture.
Imai
Y
Y
Komori
M
M
Nakamura
M
M
Kusunose
E
E
Satouchi
K
K
Kusunose
M
M
D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D006900:Hydroxylation; D007848:Laurates / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D012694:Serine / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013912:Threonine / Q000737:chemistry*; D014633:Valine / Q000737:chemistry*
D005227:Fatty Acids; D007848:Laurates; D013912:Threonine; D012694:Serine; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D014633:Valine