B. Amarneh et al., FUNCTIONAL DOMAINS OF HUMAN AROMATASE CYTOCHROME-P450 CHARACTERIZED BY LINEAR ALIGNMENT AND SITE-DIRECTED MUTAGENESIS, Molecular endocrinology, 7(12), 1993, pp. 1617-1624
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 be
en 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 tha
t although there is a similarity of overall structure in cytochromes P
450, 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 com
parison of the alignments of P450BM3 and P450cam, generated from their
three-dimensional structure, and have made mutations in regions we be
lieve to be involved in substrate recognition at the solvent surface a
nd orientation in the heme pocket. We have mutated F116 and F134 to de
termine if they are present in the home pocket, and Q225 and L228 to d
etermine if they are a part of the substrate recognition loop. Althoug
h F116E is essentially inactive and may be a folding mutant or may inh
ibit reductase binding, F134E is more active than the wild type and ma
y 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 h
ave also examined the effects of sequential deletions at the N-terminu
s of this enzyme to determine the structural requirements for activity
in this region. Although the -10 deletion retains full activity, ther
e is little activity in the -20 mutation, implying that the region bet
ween residues 10 and 20 is critical for targeting the protein or is re
quired for conformational integrity. Also, T14A shows a change in mobi
lity, as determined by sodium dodecyl sulfatepolyacrylamide gel electr
ophoresis, indicating its role as part of a glycosylation signal in P4
50arom. Finally, we have completed our series of mutations of residues
in the I-helix involved in oxygen activaton. As has been shown for P4
50cam, the conserved, threonine, which in P450arom is T310, is believe
d to bond to molecular oxygen and facilitate its activation; when muta
ted to alanine it is essentially inactive, although T310S shows activi
ty similar to that of the wild type. Mutations at D309, which is thoug
ht to reprotonate the threonine after oxygen activation, also cause a
decrease in activity, but do not completely inactivate the enzyme; thu
s, the threonine may alternatively abstract a hydrogen from some other
nearby source or from water.