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

(1)携带大鼠肝脏P450 cDNA的酿酒酵母在卤代乙烷脱卤中的高效性：肝脏P450是单加氧酶，利用NADPH的O_2和电子，肝脏P450具有数千种有毒有机底物，作为解毒酶帮助化学物质从体内排出。携带大鼠肝脏P450 1A2的酵母有效地降解三氯乙烯、五氯乙烷和六氯乙烷。底物结合表面和血红素末端位点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催化降解数千种化学物质，该方法有望用于环境污染物的化学定向降解。(2)NO合酶加氧酶结构域-P450 BM 3还原酶结构域嵌合酶的分子开关：NO合酶由一个具有P450样血红素活性位点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， ...更多信息 转运受Ca ^2 +/钙调蛋白控制。P450 BM 3也由P450加氧酶结构域和还原酶结构域组成，但催化和电子传递不受Ca ^2 +/钙调蛋白控制。为了构建具有分子开关系统的新型酶，我们构建了由NO合酶加氧酶结构域和P450 BM 3还原酶结构域组成的嵌合蛋白。新型嵌合酶的NO生成活性、底物结合和电子传递等功能均受Ca ^2 +/钙调素的调控。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提供了启示。(3)神经元型一氧化氮合酶的偶氮还原：一氧化氮合酶催化从L-Arg形成NO。该酶能有效地催化偶氮化合物的分解，而偶氮化合物是环境污染物和致癌物。这种分解是由Ca ^2 +/钙调蛋白作为分子开关控制的。因此，我们发现NO合酶在特定条件下可以降解环境中的化学物质。少

(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