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Peroxygenase P450 as a Novel Bio-material

过氧化酶 P450 作为一种新型生物材料

基本信息

批准号：
14580616
负责人：
SHIRO Yoshitsugu
金额：
$ 2.24万
依托单位：
RIKEN (2003-2004)Osaka City University (2002)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

Our research aim in this project was to understand the relationship of function and structure of cytochrome P450Bsβ from Bacillus subtilis, which can catalyze the hydroxylation of α- and β-positions of fatty acids using hydrogen peroxide H_2O_2 as an oxygen donor. We succeeded in making the single crystal and afterward in determining the structure (2.1Å resolution) of the ferric (Fe^<3+>) enzyme in the presence of a substrate (palimitic acid). On the basis of the structure, we prepared some mutants, and measured their enzymatic activities and spectral properties, to reveal roles of the mutated residues in the enzymatic functions (specificities in the substrate recognition, the catalytic reactions, the reaction sites, etc.). Then we proposed the molecular mechanism of the reaction catalyzed by P450Bsβ. In the mechanism, Arg242 binds with the carboxylate of the substrate fatty acid, which acts as a general acid-base catalyst in the peroxide O-O bond cleavage. The CO complex of the substrate-bound enzyme was determined. It was found that Phe79 was moved, but others were not upon the CO binding to the ferrous iron. To obtain a crystal of this enzyme in the substrate-free form, we examined the purification procedures for removal of the bound substrate by gel-filteration or by the product formation with H_2O_2. But we have not yet obtained the crystal of the substrate-free enzyme. In pursuing the structural determination of the short-lived reaction intermediate (possibly the Fe^<5+>=O state), we tried to stabilize the oxy complex of the enzyme in solution state, and then to inject two electrons with the X-ray reduction technique. We found the partial formation of the oxy complex. Finally, we discussed details of the reaction mechanism of heme-containing enzymes which utilize hydrogen peroxide.

本课题的研究目的是了解枯草芽孢杆菌细胞色素P450 Bs β的功能与结构的关系，该细胞色素P450 Bs β能以过氧化氢H_2O_2为氧供体催化脂肪酸的α-和β-位羟基化。我们成功地制造了单晶，然后在底物（棕榈酸）存在的情况下确定了铁（Fe^<3+>）酶的结构（分辨率为2.1 nm）。在此基础上，制备了突变体，测定了其酶活性和光谱性质，揭示了突变残基在酶功能（底物识别特异性、催化反应特异性、反应位点特异性等）中的作用。并对P450 Bs β催化反应的分子机理进行了初步探讨。在该机制中，Arg 242与底物脂肪酸的羧酸盐结合，其在过氧化物O-O键断裂中充当一般的酸碱催化剂。测定了底物结合酶的CO复合物。结果表明，当CO与亚铁离子结合时，Phe 79发生了移动，而其它的则没有发生移动。为了获得无底物形式的这种酶的晶体，我们研究了通过凝胶过滤或通过用H_2O_2形成产物来去除结合底物的纯化程序。但是我们还没有得到无底物酶的晶体。为了确定短寿命反应中间体（可能是Fe^<5+>=O态）的结构，我们试图在溶液状态下稳定酶的氧合物，然后用X射线还原技术注入两个电子。我们发现了氧合物的部分形成。最后，我们讨论了含血红素的酶，利用过氧化氢的反应机理的细节。