Functional domains of human aromatase cytochrome P450 characterized by linear alignment and site-directed mutagenesis.
Functional domains of human aromatase cytochrome P450 characterized by linear alignment and site-directed mutagenesis.
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DOI：
10.1210/mend.7.12.8145767
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发表时间：
1993-12
影响因子：
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通讯作者：
B. Amarneh;C. Corbin;J. Peterson;E. Simpson;S. Graham-Lorence
中科院分区：
文献类型：
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作者：
B. Amarneh;C. Corbin;J. Peterson;E. Simpson;S. Graham-Lorence
用序列比对和定点突变的方法，研究了芳香酶细胞色素P450(P450arom，CYP19基因产物)的功能与结构的关系。将P450arom的序列与已确定其三维结构的两种可溶性细菌细胞色素P450亚型(P450BM3和P450cam)进行了比较。从这一比较中，似乎表明尽管细胞色素P450在总体结构上有相似之处，但在参与底物识别和底物结合的区域有足够显著的差异，因此必须对被认为涉及的残基进行测试，即使是在已知的结构中。考虑到这一点，我们已经产生了P450arom的详细比对，包括根据比较P450BM3和P450cam的三维结构产生的P450BM3和P450cam的比对，包括假设的α-螺旋和β-片断的定义，并在我们认为参与底物识别的区域在溶剂表面和血红素口袋中的取向上进行了突变。我们突变了F116和F134以确定它们是否存在于血红素口袋中，并突变了Q225和L228以确定它们是否为底物识别环的一部分。虽然F116E基本上是无活性的，可能是折叠突变体或抑制还原酶结合，但F134E比野生型更有活性，可能位于血红素口袋中，有利于雄烯二酮从C2中提取氢。Q225和L228的突变也会导致表观Km和最大速度的预期变化。
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)