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价物的重要来源。作为s -腺苷甲硫氨酸（SAM），甲硫氨酸末端甲基在体内广泛参与转甲基化反应，乙基甘氨酸部分在快速增殖的细胞中参与多胺生物合成。对蛋氨酸代谢命运的兴趣是由许多人类癌细胞系依赖蛋氨酸的观察引起的，甲基硫腺苷（MTA）是SAM代谢的产物，是多胺生物合成的有效反馈抑制剂，它反过来阻止DNA复制并阻止细胞周期的延续。有证据表明，在依赖蛋氨酸的癌细胞系中，负责将S-OH3基团从MTA再循环为蛋氨酸的蛋氨酸回收途径存在缺陷。

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

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

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