Key amino acid residues required for aryl migration catalysed by the cytochrome P450 2-hydroxyisoflavanone synthase.
Key amino acid residues required for aryl migration catalysed by the cytochrome P450 2-hydroxyisoflavanone synthase.
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DOI：
10.1046/j.1365-313x.2002.01378.x
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发表时间：
2002-09
期刊：
影响因子：
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通讯作者：
Y. Sawada;K. Kinoshita;T. Akashi;T. Aoki;S. Ayabe
中科院分区：
文献类型：
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作者：
Y. Sawada;K. Kinoshita;T. Akashi;T. Aoki;S. Ayabe
异黄酮主要分布于豆科植物中，在寄主植物与生物环境的相互作用中起着重要作用。膳食中的异黄酮也作为植物雌激素对人体健康有益。在植物细胞中，细胞色素P450的CYP 93 C亚家族构成了植物细胞骨架。该反应由黄烷酮分子在C-2处的羟基化和分子内1，2-芳基从C-2迁移到C-3以产生2-羟基异黄烷酮组成。在这项研究中，与CYP 93家族P450的氨基酸序列的比对和计算机生成的推定的蛋白质的立体结构的帮助下，在CYP 93 C2的关键氨基酸残基的候选人负责的独特的芳基迁移2-羟基异黄烷酮合酶反应进行了鉴定。制备表达CYP 93 C2突变蛋白的重组酵母细胞微粒体，并检测其催化活性。与突变体的反应，其中Ser 310在中心的I-螺旋被转化为Thr产生增加的形成3-羟基黄烷酮，副产物的2-羟基异黄烷酮合酶反应，除了主要的β-羟黄烷酮产品。更引人注目的是，β-折叠1-4末端的Lys 375被Thr取代的突变体仅产生3-羟基黄烷酮，不再产生β-羟基黄烷酮。讨论了这些氨基酸残基在异黄酮生物合成的催化和进化中的作用。
Isoflavonoids are distributed predominantly in leguminous plants, and play pivotal roles in the interaction of host plants with biological environments. Isoflavones in the diet also have beneficial effects on human health as phytoestrogens. The isoflavonoid skeleton is constructed by the CYP93C subfamily of cytochrome P450s in plant cells. The reaction consists of hydroxylation of the flavanone molecule at C-2 and an intramolecular 1,2-aryl migration from C-2 to C-3 to yield 2-hydroxyisoflavanone. In this study, with the aid of alignment of amino acid sequences of CYP93 family P450s and a computer-generated putative stereo structure of the protein, candidates for key amino acid residues in CYP93C2 responsible for the unique aryl migration in 2-hydroxyisoflavanone synthase reaction were identified. Microsomes of recombinant yeast cells expressing mutant proteins of CYP93C2 were prepared, and their catalytic activities tested. The reaction with the mutant in which Ser 310 in the centre of the I-helix was converted to Thr yielded increased formation of 3-hydroxyflavanone, a by-product of the 2-hydroxyisoflavanone synthase reaction, in addition to the major isoflavonoid product. More dramatically, the mutant in which Lys 375 in the end of beta-sheet 1-4 was replaced with Thr produced only 3-hydroxyflavanone and did not yield the isoflavonoid any longer. The roles of these amino acid residues in the catalysis and evolution of isoflavonoid biosynthesis are discussed.