Structural Determinants of Aromatase Cytochrome P450 Inhibition in Substrate Recognition Site-1

0888-8809/02/$15.00/0 Printed in U.S.A. Molecular Endocrinology 16(7):1456–1468 Copyright © 2002 by The Endocrine Society Structural Determinants of Aromatase Cytochrome P450 Inhibition in Substrate Recognition Site-1 ALAN CONLEY, SAMANTHA MAPES, C. JO CORBIN, DOUGLAS GREGER*, AND SANDRA GRAHAM† Department of Population Health and Reproduction, University of California School of Veterinary Medicine (A.C., S.M., C.J.C., D.G.), Davis, California 95616; and Department of Biochemistry, University of Texas Southwestern Medical Center (S.G.), Dallas, Texas 75235 The porcine gonadal form of aromatase cytochrome P450 (P450arom) exhibits higher sensitivity to inhibition by the imidazole, etomidate, than the placental isozyme. The residue(s) responsible for this functional difference was mapped using chimeragenesis and point mutation analysis of the placental isozyme, and the kinetic analysis was conducted on native and mutant enzymes after overexpression in insect cells. The etomidate sensitivity of the placental isozyme was markedly increased by substitution of the predicted substrate recognition site-1 (SRS-1) and essentially reproduced that of the gonadal isozyme by substitution of SRS-1 and the predicted B helix. A single isoleucine (I) to methionine (M) substitution at position 133 of the placental isozyme (I133M) was proven to be the critical residue within SRS-1. Residue 133 is located in the Bⴕ-C loop and has been shown to be equally important in other steroid-metabolizing P450s. Single point mutations (including residues 110, 114, 120, 128, 137, and combinations thereof among others) and mutation of the entire B and C helixes were without marked effect on etomidate inhibitory sensitivity. The same mutation (I133M) introduced into human P450arom also markedly increased etomidate sensitivity. Mutation of Ile133 to either alanine (I133A) or tyrosine (I133Y) decreased apparent enzyme activity, but the I133A mutant was sensitive to etomidate inhibition, suggesting that it is Ile133 that decreases etomidate binding rather than Met133 increasing enzyme sensitivity. Androstenedione turnover and affinity were similar for the I133M mutant and the native placental isozyme. These data suggest that Ile133 is a contact residue in SRS-1 of P450arom, emphasize the functional conservation that exists in SRS-1 of a number of steroid-hydroxylating P450 enzymes, and suggest that substrate and inhibitor binding are dependent on different contact points to varying degrees. (Molecular Endocrinology 16: 1456–1468, 2002) E matase inhibitors have been used successfully for this (12) as well as a variety of other conditions (13–15). The potency of third generation aromatase inhibitors coupled with the possibility that they may be used more widely and that therapies may be prolonged has lead to concern over undesirable side-effects (16). Structural models of P450s offer the promise of explaining and predicting substrate preferences and inhibitor specificities (17) as well as enabling the design of drugs that effectively and specifically modulate P450 function with minimal side-effects (18). The solved structures of several bacterial P450s form the backbone of most homology models (19) because microsomal P450s have been particularly difficult to crystallize. Mutagenesis has been an essential tool in testing and refining these to represent mammalian P450 isozymes (20), but it is still unclear what basic structural differences dictate the catalytic differences among them, substrate preferences and stereospecificity for instance. Two basic approaches have been taken in designing mutations. One identifies and targets residues based on the model itself, and generally a loss of function upon mutation is taken as evidence of the accuracy of the model. This has been the strategy used in almost all structure-function studies of STROGENS ARE synthesized from androgens by the enzyme known as aromatase cytochrome P450 (P450arom). This enzyme is expressed in a number of tissues in the body, including male and female gonads, brain, placenta, and adipose tissue among others (1, 2). Expression is regulated in a distinct tissue-specific fashion and is critical to normal sexual development and subsequent fertility (3). However, it is becoming increasingly clear that either the lack of expression or overexpression of P450arom can lead to disease in a wide variety of tissues (4). Breast cancer is believed to develop and progress as a result of the local production of estrogen from the aberrant expression of P450arom in adipose stromal cells (5). As a consequence, great effort has been expended in devising pharmacological inhibitors of P450arom (6–9), and some of the most recently developed, the socalled third generation imidazole and steroidal inhibitors, rank among the most potent and specific known for any P450. Early results with these compounds have been so encouraging that prophylactic treatment has been considered for women at high risk (10). Aberrant expression of P450arom has also been implicated in other diseases, such as endometriosis (11), and aroAbbreviation: P450arom, Aromatase cytochrome P450. 1456 Conley et al. • Aromatase Cytochrome P450 Inhibition in SRS-1 P450arom (21–28). These studies have relied on expression by transient transfection or in stably transfected cell lines in part because of difficulties in overexpressing P450arom in bacteria, which has been successful only after deletion of amino-terminal residues (29, 30). An alternative approach relies on catalytic or functional differences between closely related isozymes, from different species or within a species (31, 32). Interchanging nonidentical residues between different isozymes and thereby mapping function empirically has been used extensively and with notable success to gain significant insights into the substrate binding pocket of a number of different P450s from the 2A, 3A, 2B, and 2C families among others (20). Gotoh (33) used a computer analysis of the CYP2C family of P450s to identify six major domains, which he designated substrate recognition sites (SRS). These, he postulated, were involved in substrate binding and accounted for differences in substrate specificities between isozymes within the family. The results of chimeric enzyme and site-directed mutagenesis studies of SRSs in 3A4, 2B, and other P450 isozymes (34–39) generally support this interpretation, although not precluding the likely importance of residues outside these regions (40, 41). To our knowledge, this approach, using catalytic differences among isozymes to design mutants, has never been used in structure-function studies of P450arom despite the fact that over a dozen mammalian P450arom enzymes have been cloned (2). The apparent functional conservation among aromatases from different species, even from different vertebrate classes, has left little in the way of catalytic differences that could be usefully mapped. However, the existence of multiple (...truncated)