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

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(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