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Structural and functional analysis of anti-tumor microbial enzymes and their application for development of next generation anti-tutor enzymes.

抗肿瘤微生物酶的结构和功能分析及其在开发下一代抗导师酶中的应用。

基本信息

批准号：
18580076
负责人：
INAGAKI Kenji
金额：
$ 2.49万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

A highly potent recombinant L-methionine γ-lyase from Pseudomonas putida (MGL_Pp. EC 4.4.1.11) has been characterized physicochemically and pharmacokinetically in vivo and in vitro as a potent anticancer agent that can deplete L-methionine from plasma. The detailed structure of MGL_Pp and the function of the active site residue have so far not been cleared to improve as a next generation antitumor enzyme. We demonstrated that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method at 1.8A resolution. Detailed information of the overall structure of MGL_Pp supplied clear pictures of the N-terminal domain and the substrate-and PLP-binding pockets. It was found that a hydrogen bond network was formed around a cofactor pyridoxal 5'-phosphate in the MGL active site, which is specific for MGLs. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug. The 3D structure of MGL_Pp suggested that Cys116 might be involved in the hydrogen bond network at the active site. We found that Cys116 plays an important role in the γ-elimination reaction of L-methionine and for the substrate recognition in the MGLs. We also discovered that the substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a change of the substrate specificity, suggesting that the His residue may be directly involved in the γ-elimination reaction. With a low purity, it was found that MGL_Pp was degraded between Cys49-Phe50 and the degraded enzyme lost the activity. Conjugation of MGL_Pp with polyethylene glycol (PEG) will importantly reduce proteolysis. Recently, the complete nucleotide sequence of the linear chromosome of S. avermitilis has been determined. The gene encoding L-methionine γ-lyase from Streptomyces avermitilis was cloned and expressed in Escherichia coli to characterize the enzymological properties of the gene product. MGL_Sa. for the first time over the world.

从恶臭假单胞菌（Pseudomonasputida，MGL_Pp. EC 4.4.1.11）已经在体内和体外被表征为可以从血浆中消耗L-甲硫氨酸的有效抗癌剂。MGL_Pp的详细结构和活性位点残基的功能迄今尚未明确，以作为下一代抗肿瘤酶进行改进。我们证明，MGL_Pp的三维结构已通过分子置换方法以1.8A的分辨率完全解决。MGL_Pp的整体结构的详细信息提供了N-末端结构域和底物-和PLP-结合口袋的清晰图片。发现在MGL活性位点中的辅因子吡哆醛5 '-磷酸周围形成氢键网络，这对MGL是特异性的。详细的结构将有助于MGL_Pp作为抗癌药物的开发。MGL_Pp的三维结构表明Cys 116可能参与了活性中心的氢键网络。我们发现Cys 116在L-甲硫氨酸的γ-消除反应和MGL中的底物识别中起重要作用。我们还发现，Cys 116取代His导致对L-半胱氨酸的活性显著增加，并且改变了底物特异性，这表明His残基可能直接参与γ-消除反应。结果表明，MGL_Pp在Cys 49-Phe 50之间发生降解，降解后的酶失去活性，纯度较低。MGL_Pp与聚乙二醇（PEG）的缀合将重要地减少蛋白水解。最近，已测定了该菌线状染色体的全序列。已测定阿维菌素。克隆了阿维链霉菌L-甲硫氨酸γ-裂解酶基因，并在大肠杆菌中表达，研究了其酶学性质。MGL_Sa.第一次在世界各地。