Functional domains of human aromatase cytochrome P450 characterized by linear alignment and site-directed mutagenesis.
The relationship of function to structure of aromatase cytochrome P450 (P450arom; the product of the CYP19 gene) has been examined by means of sequence alignment and site-directed mutagenesis. Comparison has been made between the sequence of P450arom and the two soluble bacterial cytochrome P450 isoforms, whose three-dimensional structure has been determined (P450BM3 and P450cam). From this comparison, it appears that although there is a similarity of overall structure in cytochromes P450, there is enough significant difference in the regions involved in substrate recognition and substrate binding that residues believed to be involved, even in the known structures, must be tested. With this in mind, we have generated a detailed alignment of P450arom, including the definition of putative alpha-helices and beta-sheets based on comparison of the alignments of P450BM3 and P450cam, generated from their three-dimensional structure, and have made mutations in regions we believe to be involved in substrate recognition at the solvent surface and orientation in the heme pocket. We have mutated F116 and F134 to determine if they are present in the heme pocket, and Q225 and L228 to determine if they are a part of the substrate recognition loop. Although F116E is essentially inactive and may be a folding mutant or may inhibit reductase binding, F134E is more active than the wild type and may be located in the heme pocket facilitating the hydrogen abstraction from C2 of androstenedione. Mutations at Q225 and L228 also result in the anticipated changes in the apparent Km and maximum velocity.(ABSTRACT TRUNCATED AT 250 WORDS)
Amarneh
B
B
Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Obstetrics-Gynecology, University of Texas Southwestern Medical Center, Dallas 75235-9051.
Corbin
C J
CJ
Peterson
J A
JA
Simpson
E R
ER
Graham-Lorence
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001141:Aromatase / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D001141:Aromatase