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TOXIN-DIRECTED MOLECULAR CONVERSION SYSTEM WITH YEAST HARBORING HIGHLY ACTIVATED P450 ENZYME BY ARTIFICIAL MUTAGENESIS

通过人工诱变构建含有高度活化 P450 酶的酵母的毒素导向分子转化系统

基本信息

批准号：
09480130
负责人：
SHIMIZU Toru
金额：
$ 8.13万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

项目摘要

(1) High utility of Saccharomyces cerevisiae harboring rat liver P450 cDNA in haloethanes dehalogenations : Liver P450 is monooxygenase and uses O_2 and electrons from NADPH.Liver P450s have thousands of toxic organic substrates and work as detoxic enzyme to help chemicals to be eliminated from the body. Yeast harboring rat liver P450 1A2 efficiently degraded trichloroethylene, pentachloroethane and hexachloroethane. Mutations on the substrate binding surface and heme distal site enormously enhanced the catalytic activity toward those haloethanes. Since liver P450 catalyze degradation of thousands of chemicals, this method is promising for chemical-directed degradation of environmental pollutants.(2) Molecular switch of NO synthase oxygenase domain-P450BM3 reductase domain chimeric enzyme : NO synthase is composed of an oxygenase domain with P450-like heme active site and a reductase domain which is similar to NADPH-P450 reductase and the NO formation activity and interdomain electron … More transfer is controlled by Ca^<2+>/calmodulin. P450BM3 is also composed of the P450 oxygenase domain and the reductase domain but the catalysis and the electron transfer are not controlled by Ca^<2+>/calmodulin. In order to construct a novel enzyme with molecular switch system, we generated a chimeric protein composed of the NO synthase oxygenase domain and the P450BM3 reductase domain. The new chimeric enzyme functions including NO formation activity, substrate binding and electron transfer were controlled by Ca^<2+>/calmodulin. Thus, this protein eaxgineering approach sheds light for application of the enzymatic system with molecular switch to environmental degradation.(3) Azo reduction of neuronal nitric oxide synthase :Nitric oxide synthase catalyzes NO formation from L-Arg. This enzyme efficiently catalyzed the decomposition of one of azo compounds, which are often environmental pollutamts and carcinogen. This decomposition was controlled with Ca^<2+>/calmodulin as a molecular switch. Therefore, we found that NO synthase could work to degrade environmental chemical under specific conditions. Less

(1)携带大鼠肝脏P450基因的酿酒酵母在卤代乙烷脱卤化中的高效利用：肝脏P450是单加氧酶，利用NADPH产生的氧和电子。肝脏P450含有数千种有毒的有机底物，作为解毒酶帮助化学物质从体内排出。携带大鼠肝脏P450 1A2的酵母能有效降解三氯乙烯、五氯乙烷和六氯乙烷。底物结合表面和血红素远端位置的突变极大地增强了对这些卤代乙烷的催化活性。由于肝脏P450催化降解数以千计的化学物质，这种方法在化学定向降解环境污染物方面很有前途。(2)NO合成酶的分子开关-P450BM3还原酶域的嵌合酶：NO合成酶是由一个具有类似P450的血红素活性中心的加氧酶结构域和一个类似于NADPH-P450还原酶的还原酶结构域组成的，它的NO生成活性和域间电子…更多的转运是由钙离子/钙调素控制的。P450BM3也由P450加氧酶结构域和还原酶结构域组成，但其催化和电子传递不受Ca~(2+)/CaM调控。为了构建具有分子开关系统的新型酶，我们构建了一个由一氧化氮合酶加氧酶结构域和P450BM3还原酶结构域组成的嵌合蛋白。新的嵌合酶功能包括NO形成活性、底物结合和电子传递受钙调素/钙调素调控。因此，这种蛋白质工程方法为具有分子开关的酶系统在环境降解中的应用奠定了基础。(3)神经元型一氧化氮合酶的偶氮还原：一氧化氮合酶催化L-精氨酸生成一氧化氮。这种酶有效地催化了一种偶氮化合物的分解，这些偶氮化合物通常是环境污染物和致癌物质。这种分解是由Ca~(2+)和钙调素作为分子开关控制的。因此，我们发现在特定的条件下，没有合酶能够降解环境中的化学物质。较少