A Novel CO-producing Metalloenzyme

一种新型联产金属酶

• 批准号: 7060039
• 负责人: Thomas Charles Pochapsky
• 金额: $ 20.43万
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7060039
• 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）：蛋氨酸是活细胞中烷基化等效物的重要来源。作为S-腺苷甲氨酸（SAM），甲基蛋氨酸的末端甲基在体内的多种跨甲基化反应中使用，在快速增殖的细胞中，乙基甘氨酸部分在多胺生物合成中馈入聚酰胺生物合成。通过观察到，许多人类癌细胞系都是蛋氨酸依赖性的，并且甲基噻吩腺苷（MTA）是SAM代谢的产物，是一种有效的多胺生物合成反馈抑制剂，这反过来避免DNA复制和持续细胞周期。有证据表明，在蛋氨酸依赖性的癌细胞系中，蛋氨酸拯救途径是从MTA回收S-OH3组的，该途径是从MTA回收S-OH3组。 该提案描述了对来自克雷伯氏菌肺炎细菌的蛋氨酸挽救途径，呼吸酶dioxygenase酶（ARD）的蛋氨酸挽救途径的显着属酶的结构和功能的深入研究。 ARD可以根据与酶活性位点结合的金属离子的相同底物催化两种不同的反应。含有单个Ni+2离子的ARD催化了蛋氨酸拯救途径中的一种晚期中间体的氧化性裂解，进入β-甲基硫代二丙酸，富甲酸甲酸酯，一氧化碳和一氧化碳（CO）中的氧化中间体（CO）。如果Fe+2离子代替镍（ARD'），则导致甲酸甲酸甲酸酯和酮酸前体的甲硫氨酸前体，Alpha-Keto-Gamma--甲基硫硫酸酯的催化剂是催化的。 NMR方法最近已解决了NI结合的ARD的结构，机械研究为通过ARD催化的反应的根本机制提供了证据。 拟议工作的一般目的包括：（1）确定ARD'的溶液结构，以及对导致其不同活性的ARD和ARD之间的结构差异的分析。将进行稳定酶 - 基底络合物的位置定向诱变，酶活性和动力学测量和NMR研究。 （2）来自两个真核生物，大米（Oriza sativa）和人类（Homo Sapiens）的ARD同源物的表达，隔离，纯化和表征。特别是，将寻求体外和体内ARD活性的证据（即CO生产），因为现在有大量证据表明CO可以充当信号分子和凋亡的抑制剂（程序性细胞死亡）。

项目成果

(1)H, (13)C and (15)N chemical shift assignments of an enolase-phosphatase, E1, from Klebsiella oxytoca.

来自产酸克雷伯氏菌的烯醇化磷酸酶 E1 的 (1)H、(13)C 和 (15)N 化学位移归属。

• DOI: 10.1007/s10858-005-1128-2
• 发表时间: 2004
• 期刊: Journal of biomolecular NMR
• 影响因子: 2.7
• 作者: Kostic,Milka;Pochapsky,ThomasC
• 通讯作者: Pochapsky,ThomasC

The immediate-early ethylene response gene OsARD1 encodes an acireductone dioxygenase involved in recycling of the ethylene precursor S-adenosylmethionine.

• DOI: 10.1111/j.1365-313x.2005.02564.x
• 发表时间: 2005-11
• 期刊: The Plant journal : for cell and molecular biology
• 作者: M. Sauter;René Lorbiecke;B. Ouyang;T. Pochapsky;Guillaume Rzewuski

Analogs of 1-phosphonooxy-2,2-dihydroxy-3-oxo-5-(methylthio)pentane, an acyclic intermediate in the methionine salvage pathway: a new preparation and characterization of activity with E1 enolase/phosphatase from Klebsiella oxytoca.

1-膦酰氧基-2,2-二羟基-3-氧代-5-(甲硫基)戊烷的类似物，甲硫氨酸补救途径中的无环中间体：来自产酸克雷伯氏菌的 E1 烯醇酶/磷酸酶的新制备和活性表征。

• DOI: 10.1016/j.bmc.2004.05.002
• 发表时间: 2004
• 期刊: Bioorganic & medicinal chemistry
• 影响因子: 3.5
• 作者: Zhang,Yalin;Heinsen,MelissaH;Kostic,Milka;Pagani,GinaM;Riera,ThomasV;Perovic,Iva;Hedstrom,Lizbeth;Snider,BarryB;Pochapsky,ThomasC
• 通讯作者: Pochapsky,ThomasC