Structure-function analysis of CYP2A10 and CYP2A11, P450 cytochromes that differ in only eight amino acids but have strikingly different activities toward testosterone and coumarin.
Cytochrome P450 NMa, which was first identified in this laboratory in rabbit nasal microsomes, is now known to represent two distinct gene products, P450s 2A10 and 2A11. In the present study, chimeric and site-directed mutants of 2A11 were constructed to determine which of the eight different amino acid residues are responsible for the much greater activity of 2A10 toward coumarin and testosterone. Mutation of Arg62 and Asp63 of 2A11 to the corresponding residues in 2A10, or mutation of Thr120 to Ser, as found in 2A10, did not change the activities. However, mutation of Arg62, Asp63, Gln104, Ala117, and Thr120 of 2A11 to the corresponding residues in 2A10 resulted in a protein that is as active as 2A10 in coumarin hydroxylation and approximately half as active as 2A10 in androstenedione formation. Mutation of Arg372 in 2A11 to His, as found in 2A10, resulted in a significant increase in the rate of hydroxylation of testosterone, but not of coumarin. Our findings indicate that the identify of the amino acid at position 104 and/or 117 is important for activity with testosterone and for regioselectivity at the 17 position, as well as for optimal activity with coumarin. In contrast, the identity of the residue at position 372 is important for optimal activity with testosterone but not the regioselectivity at the 17 position and does not influence the activity with coumarin.
Ding
X
X
Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor.
Peng
H M
HM
Coon
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D003374:Coumarins / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D017931:DNA Primers; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009666:Nose; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism*
D003374:Coumarins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; C030123:coumarin; D001189:Aryl Hydrocarbon Hydroxylases; C082663:CYP2A10 protein, Oryctolagus cuniculus; C082665:CYP2A11 protein, Oryctolagus cuniculus; D000072467:Cytochrome P450 Family 2