Reciprocal size-effect relationship of the key residues in determining regio- and stereospecificities of DHEA hydroxylase activity in P450 2a5.
Collectively, the P450 2a4/2a5 system hyrdoxylates DHEA in at least three positions (7alpha, 7beta, and 2alpha). An individual P450, however, exhibits high specificity to one of these products. Using site-directed mutagenesis of mP450 2a5 from the wild mouse Mus minutoides and bacterial expression, we have associated the function of residues 117, 209, and 481 with the respective specificity observed in each P450. Ala at position 117 determines the 7beta-hydroxylase activity, whereas Val at this position defines the 2alpha-hydroxylase activity. Leu at position 209 is essential for high DHEA 7alpha-hydroxylase activity. The substitutions of residue 481 with various hydrophobic amino acids elicited a profound alteration of the specific hydroxylation rates, but did not influence the regio- and stereospecificities at either of the three positions of DHEA. The alterations caused by residue 481 also depended on the residue identity at position 117 or 209. The results indicate that the sizes of several key residues obey a concerted reciprocal relationship whereby the substrate pocket of the P450s adjusts to accommodate DHEA. A limited molecular modeling study successfully correlates DHEA binding to experimental DHEA hydroxylase activities for a series of mutants at key positions.
Uno
T
T
Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Mitchell
E
E
Aida
K
K
Lambert
M H
MH
Darden
T A
TA
Pedersen
L G
LG
Negishi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D003001:Cloning, Molecular; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009115:Muridae; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6