Altering the regiospecificity of androstenedione hydroxylase activity in P450s 2a-4/5 by a mutation of the residue at position 481.
Mouse P450 2a-5 (coumarin 7-hydroxylase) acquires androstenedione (AD) hydroxylase activity by substituting Phe at position 209 with Asn. However, this mutant P450 2a-5 (F209N) and the corresponding mutant P450 2a-4 (L209N) exhibit different regiospecificites of androstenedione (AD) hydroxylase activity. While the former mutant catalyzes both AD 15 alpha- and 7 alpha-hydroxylase activities at similar rates, the latter mutant maintains the original high specificity of AD 15 alpha-hydroxylase activity. The AD hydroxylase activities in chimeric enzymes of the mutants L209N and F209N show that the regiospecificites are determined by the carboxy-terminal halves of the P450 molecules. Mutations at each of the four different residues within the carboxy-terminal halves indicate that the differences in regiospecificity are determined by the Val/Ala mutation at position 481. As the size of the hydrophobic amino acid at position 481 becomes larger (Ala < Val < Ile), the regiospecificities toward the C15 position of the AD molecule are dramatically increased. The regiospecificity is also increased by placing positively-charged Arg at position 481, although the remaining 15 alpha-hydroxylase activity in this mutant is considerably lower than the other P450s. The results indicate that the size of the residue at position 481 is a key factor in regulating the regiospecificity of AD hydroxylase activity in the P450s. Modeling AD in the substrate-heme pocket of bacterial P450 101A provided further support that residue 481 may reside near the steroid molecule so as to possibly affect the AD hydroxylase activity.
Iwasaki
M
M
Pharmacogenetics Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Darden
T A
TA
Pedersen
L G
LG
Negishi
M
M
D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6; C050356:steroid 15-alpha-hydroxylase