Functional domains of human aromatase cytochrome P450 characterized by linear alignment and site-directed mutagenesis.

Abstract

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)

Active site acidic residues and structural analysis of modelled human aromatase: A potential drug target for breast cancer

“…This is in agreement with earlier site directed mutagenesis studies which showed decrease in enzymatic activity with androgens on mutating this residue [6,42]. However, the SAS values of D309 show significant decrease only in the presence of steroidal substrates implying that the increasing burial of this residue (D309) due to the steroids would lead to favorable interactions between the charged carboxyl groups and buried polar groups in the enzyme.…”

“…Earlier studies by Amarneh et al [6] implied that the residue R365 in the K-helix is buried internally in a hydrophobic region. This residue is found to be highly buried in the model with moderately polar environment, which may be essentially due to the presence of a conserved glutamate [6].…”

“…All three reactions are stereo specific and utilize 3 mol of oxygen and 3 mol of NADPH [3][4][5]. In particular, acidic amino acid residues in the active site of human aromatase play a vital role in determining the efficacy of proton transport essential for aromatization of androgens [6]. Nevertheless, the enzymatic mechanism of estrogen formation and the role of these acidic residues is not totally understood.…”

Nonsteroidal aromatase inhibitors: Recent advances

“…103 However, subsequent resolution of two other P450 enzymes (P450 BM-3 , 104 P450 terp 105 ) led to a revision of the models, such to take into account the new structural information, and to refine the sequence alignments, that, with the use of P450 cam only, were rather uncertain in some portions of the protein. 106,107 The development of a whole protein model of aromatase was then due to Graham-Lorence et al, 108 who used it not only to describe structural characteristics of the enzyme, but also to explain the mechanism of action postulated in the hypothesis by Akhtar (Fig. 5a).…”

A three‐dimensional model of aromatase cytochrome P450

“…Following the computer-generated sequence alignment, the alignment of P450arom with the structural alignment of P450cam, P450BM-P, and P450terp was further refined by a hand alignment resulting in the alignment shown in Figure 2. Details of these alignments are discussed more fully elsewhere (Amarneh et al, 1993); however, from the structural alignments, it is apparent that the C-terminal portion of P450s starting at the I-helix is easily aligned using almost exclusively the GCG Pileup program. Contained in this half of the molecule is the structurally conserved hydrophobic I-helix, which contains the consensus sequence (A/G)-G-X-(E/D)-T in the center of the helix and which lies directly over pyrrole ring B of the heme, except in P450arom, in which the sequence is A-A-P-D-T. (This will be discussed later in the text and shown in Figure 4.)…”