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 home 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 K-m and maximum velocity. We have also examined the effects of sequential deletions at the N-terminus of this enzyme to determine the structural requirements for activity in this region. Although the -10 deletion retains full activity, there is little activity in the -20 mutation, implying that the region between residues 10 and 20 is critical for targeting the protein or is required for conformational integrity. Also, T14A shows a change in mobility, as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, indicating its role as part of a glycosylation signal in P450arom. Finally, we have completed our series of mutations of residues in the I-helix involved in oxygen activaton. As has been shown for P450cam, the conserved, threonine, which in P450arom is T310, is believed to bond to molecular oxygen and facilitate its activation; when mutated to alanine it is essentially inactive, although T310S shows activity similar to that of the wild type. Mutations at D309, which is thought to reprotonate the threonine after oxygen activation, also cause a decrease in activity, but do not completely inactivate the enzyme; thus, the threonine may alternatively abstract a hydrogen from some other nearby source or from water.