A Novel CO-producing Metalloenzyme

一种新型联产金属酶

• 批准号: 6599429
• 负责人: Thomas Charles Pochapsky
• 金额: $ 25.87万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-05 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6599429
• 关键词: Klebsiella pneumoniae; X ray crystallography; active sites; bacterial proteins; carbon monoxide; catalyst; electron spin resonance spectroscopy; enzyme activity; enzyme structure; enzyme substrate; eukaryote; metalloenzyme; methionine; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; oxygenases; protein structure function; radiotracer; rice; serial analysis of gene expression; site directed mutagenesis

DESCRIPTION (provided by applicant): Methionine is an important source of alkylating equivalents in living cells. As S-adenosylmethionine (SAM), the terminal methyl group of methionine is used in a wide array of transmethylation reactions in vivo, and the ethylglycine moiety feeds into polyamine biosynthesis in rapidly proliferating cells. Interest in the metabolic fate of methionine is raised by the observation that many human cancer cell lines are methionine dependent, and that methylthioadenosine (MTA), a product of SAM metabolism, is a potent feedback inhibitor of polyamine biosynthesis, which in turn stops DNA replication and prevents continuation of the cell cycle. There is evidence suggesting that the methionine salvage pathway, which is responsible for recycling the S-OH3 group from MTA to methionine, is defective in methionine-dependent cancer cell lines. This proposal describes an in-depth investigation into the structure and function of a remarkable metalloenzyme from the methionine salvage pathway of the bacterium Klebsiella pneumoniae, acireductone dioxygenase (ARD). ARD can catalyze two different reactions with the same substrate depending upon the metal ion that is bound to the enzyme active site. ARD containing a single Ni+2 ion catalyzes the oxidative cleavage of acireductone, an advanced intermediate in the methionine salvage pathway, into beta-methylthiopropionate, formate and carbon monoxide (CO) in an off-pathway shunt. If a Fe+2 ion is bound instead of nickel (ARD'), the on-pathway reaction leading to formate and the ketoacid precursor of methionine, alpha-keto-gamma-methylthiobutyrate is catalyzed. A structure for Ni-bound ARD has recently been solved by NMR methods, and mechanistic studies have provided evidence for a radical mechanism for the reaction catalyzed by ARD. The general aims of the proposed work include: (1) Determination of the solution structure of ARD' and an analysis of the structural differences between ARD and ARD' that lead to their different activities. Site-directed mutagenesis, enzyme activity and kinetics measurements and NMR studies of stable enzyme-substrate complexes will be performed. (2) Expression, isolation, purification and characterization of ARD homologues from two eukaryotes, rice (Oriza sativa) and humans (Homo sapiens). In particular, evidence for in-vitro and in-vivo ARD activity will be sought (i.e., CO production), since there is now considerable evidence that CO can act as a signaling molecule and an inhibitor of apoptosis (programmed cell death).

描述（由申请人提供）：甲硫氨酸是活细胞中烷基化等同物的重要来源。作为S-腺苷甲硫氨酸（SAM），甲硫氨酸的末端甲基用于体内广泛的甲基转移反应，乙基甘氨酸部分用于快速增殖细胞中的多胺生物合成。许多人类癌细胞系是蛋氨酸依赖性的，并且甲硫腺苷（MTA），SAM代谢的产物，是多胺生物合成的有效反馈抑制剂，这反过来阻止DNA复制并阻止细胞周期的延续，这引起了对蛋氨酸代谢命运的兴趣。有证据表明，甲硫氨酸补救途径，这是负责回收的S-OH 3基团从MTA的甲硫氨酸，是有缺陷的甲硫氨酸依赖性癌细胞系。 该提案描述了一个显着的金属酶的结构和功能的深入调查从蛋氨酸补救途径的细菌肺炎克雷伯氏菌，酸还原酮双加氧酶（ARD）。ARD可以催化两个不同的反应，与相同的底物取决于金属离子结合到酶的活性位点。含有单个Ni+2离子的ARD可催化甲硫氨酸补救途径中的高级中间体酸还原酮氧化裂解为途径外分流中的β-甲硫基丙酸酯、甲酸酯和一氧化碳（CO）。如果结合的是Fe+2离子而不是镍（ARD '），则催化了导致甲酸盐和甲硫氨酸的酮酸前体α-酮基-γ-甲硫基丁酸盐的途径上反应。最近已经解决了镍结合ARD的结构通过NMR方法，和机理的研究提供了证据的ARD催化的反应的自由基机制。 本文的主要工作包括：（1）确定ARD'的溶液结构，并分析ARD和ARD'的结构差异导致它们不同的活性。将进行定点诱变、酶活性和动力学测量以及稳定酶-底物复合物的NMR研究。(2)来自两种真核生物，水稻（Oriza sativa）和人类（Homo sapiens）的ARD同源物的表达、分离、纯化和表征。特别地，将寻找体外和体内ARD活性的证据（即，CO产生），因为现在有相当多的证据表明，CO可以作为信号分子和细胞凋亡（程序性细胞死亡）的抑制剂。