A Novel CO-producing Metalloenzyme
一种新型联产金属酶
基本信息
- 批准号:7060039
- 负责人:
- 金额:$ 20.43万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:Klebsiella pneumoniaeX ray crystallographyactive sitesbacterial proteinscarbon monoxidecatalystelectron spin resonance spectroscopyenzyme activityenzyme structureenzyme substrateeukaryotemetalloenzymemethioninenuclear magnetic resonance spectroscopyoxidation reduction reactionoxygenasesprotein structure functionradiotracerriceserial analysis of gene expressionsite directed mutagenesis
项目摘要
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
期刊论文数量(7)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
(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:
- 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:
- 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:
- 影响因子:0
- 作者:M. Sauter;René Lorbiecke;B. Ouyang;T. Pochapsky;Guillaume Rzewuski
- 通讯作者:M. Sauter;René Lorbiecke;B. Ouyang;T. Pochapsky;Guillaume Rzewuski
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Thomas Charles Pochapsky其他文献
Thomas Charles Pochapsky的其他文献
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{{ truncateString('Thomas Charles Pochapsky', 18)}}的其他基金
Structure and dynamics of clinically-relevant cytochrome P450 enzymes - Summer undergraduate research experience supplement
临床相关细胞色素 P450 酶的结构和动力学 - 暑期本科生研究经验补充
- 批准号:
10392567 - 财政年份:2019
- 资助金额:
$ 20.43万 - 项目类别:
Structure and dynamics of clinically-relevant cytochrome P450 enzymes
临床相关细胞色素 P450 酶的结构和动力学
- 批准号:
10297854 - 财政年份:2019
- 资助金额:
$ 20.43万 - 项目类别:
Structure and dynamics of clinically-relevant cytochrome P450 enzymes
临床相关细胞色素 P450 酶的结构和动力学
- 批准号:
10061625 - 财政年份:2019
- 资助金额:
$ 20.43万 - 项目类别:
Structure and Dynamics of Metal-Containing Proteins
含金属蛋白质的结构和动力学
- 批准号:
7924934 - 财政年份:2009
- 资助金额:
$ 20.43万 - 项目类别:
An 800 MHz NMR spectrometer for the Boston area
适用于波士顿地区的 800 MHz NMR 波谱仪
- 批准号:
6501637 - 财政年份:2003
- 资助金额:
$ 20.43万 - 项目类别:
RECONSTITUTION OF ION FERREDOXIN W/ DIAMAGNETIC METAL ION: GALLIUM PUTIDAREDOXIN
用抗磁性金属离子重建离子铁氧还蛋白:镓腐铁氧还蛋白
- 批准号:
6307603 - 财政年份:1999
- 资助金额:
$ 20.43万 - 项目类别:
RECONSTRUCION OF TWO ION FERREDOXIN W/ DIAMAGNETIC METAL ION
用抗磁性金属离子重建双离子铁氧还蛋白
- 批准号:
6118277 - 财政年份:1998
- 资助金额:
$ 20.43万 - 项目类别:
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