The Enzymology of Phosphonate Metabolism

磷酸盐代谢的酶学

• 批准号: 9113961
• 负责人: Frank M. Raushel
• 金额: $ 27.06万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-12 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9113961
• 关键词: Active Sites; Adenine; Antibiotics; Antiviral Agents; Biochemical; Carbohydrates; Carbon; Chemicals; Cleaved cell; Complex; Diphosphates; Enzymatic Biochemistry; Enzymes; Escherichia coli; Gene Cluster; Genes; Genetic; Gram-Negative Bacteria; Health; Herbicides; Human; Hydrolysis; Lyase; Metabolism; MgATP; Molecular; Multienzyme Complexes; Nucleic Acids; Nutrient; Operon; Phospholipids; Phosphorus; Prokaryotic Cells; Proteins; Reaction; Research; Ribose; Role; S-Adenosylmethionine; Scheme; Specificity; Structure; Substrate Specificity; base; direct application; enzyme mechanism; inorganic phosphate; insight; methylphosphonate; nerve agent; novel; phosphonate; protein structure; public health relevance; research study; tripolyphosphate

DESCRIPTION (provided by applicant): The broad long-term objective for the research described in this proposal is aimed towards a more comprehensive understanding of the relationship between structure and function in enzyme- catalyzed reactions. This application seeks to provide a fundamental molecular description of the factors that govern protein structure, substrate recognition, and reaction specificity. The primary focus of this proposal is directed towards the elucidation of the chemical mechanisms for the enzyme catalyzed reactions that govern the metabolism of phosphonates to phosphate. Phosphorus is an integral component of nucleic acids, carbohydrates and phospholipids. The metabolism of organophosphonates is of significant importance to human health since these compounds constitute a rapidly growing class of antibiotics, herbicides, nerve agents and antiviral drugs. However, a molecular description for the cleavage of an inactivated phosphorus-carbon bond within phosphonate substrates has not previously been elucidated, despite much effort for more than three decades. In prokaryotes the catalytic machinery for the C-P lyase reaction has been localized to the phn gene cluster. It is proposed in this application that the C-P lyase complex converts methyl phosphonate to methane and D-ribose-1,2-cyclic-phosphate-5-phosphate through the combined actions of three proteins: PhnI, PhnM, and PhnJ. The specific aims for this application are directed at determining the detailed reaction mechanisms for each of these enzymes. Phn I catalyzes the conversion of MgATP and methyl phosphonate to D-ribose-1-methylphosphonate-5-triphosphate and adenine in the presence of PhnGHL. PhnM then catalyzes the hydrolysis of D-ribose-1-methylphosphonate-5-triphosphate to D- ribose-1-methylphosphonate-5-phosphate and pyrophosphate. PhnJ then catalyzes the conversion of D-ribose-1-methylphosphonate-5-phosphate to D-ribose-1,2-cyclic-phosphate-5-phosphate and methane. The final transformation requires an [Fe4S4]-cluster and S-adenosylmethionine for catalytic activity, and thus PhnJ is a novel radical-SAM enzyme that catalyzes the cleavage of the P-C bond of phosphonates via radical-based intermediates. The proposed project will provide significantly new insights into the mechanisms and reaction diversity of the radical-SAM class of enzymes and will contribute to a greater understanding of how multi-enzyme complexes with several active sites are able to more efficiently channel products from one active site to another.

描述（由申请人提供）：本提案中描述的研究的广泛长期目标旨在更全面地了解酶催化反应中的结构与功能之间的关系。该应用旨在提供控制蛋白质结构，底物识别和反应特异性因素的基本分子描述。该提案的主要重点是阐明酶催化反应的化学机制，该反应控制磷酸盐的代谢。磷是核酸，碳水化合物和磷脂的组成部分。有机膦酸盐的代谢对人类健康至关重要，因为这些化合物构成了快速生长的抗生素，除草剂，神经剂和抗病毒药物。然而，尽管超过三十年的努力，但以前尚未阐明磷酸底物中灭活的磷碳键的分子描述。在原核生物中，C-P裂解酶反应的催化机制已定位在PHN基因簇中。在此应用中提出的是，C-P裂解酶复合物将磷酸甲酯转化为甲烷和D-核糖-1,2-磷酸磷酸磷酸5-磷酸，通过三种蛋白质的联合作用：PHNI，PHNM和PHNJ。该应用的具体目的旨在确定每种酶的详细反应机制。 PHN I催化MGATP和磷酸甲酯向D-ribose-1-甲基膦酸酯-5-三磷酸和腺嘌呤的转化。然后，PHNM催化D-核糖1-甲基膦酸酯-5-三磷酸的水解为D-核糖1-甲基膦酸5-磷酸和焦磷酸盐的水解。然后，PHNJ催化D-核糖-1-甲基膦酸酯5-磷酸向D-核糖1,2-偶然磷酸磷酸5-磷酸和甲烷的转化。最终转化需要[Fe4S4]簇和S-腺苷甲硫氨酸才能进行催化活性，因此PHNJ是一种新型的自由基SAM酶，可通过自由基中间体催化磷酸盐P-C键的裂解。拟议的项目将为激进SAM类酶的机制和反应多样性提供大量新的见解，并将有助于更深入地了解多个活性位点的多酶复合物如何能够更有效地将产品从一个活性位点传播到另一种位置。

项目成果

Discovery of a cyclic phosphodiesterase that catalyzes the sequential hydrolysis of both ester bonds to phosphorus.

• DOI: 10.1021/ja409376k
• 发表时间: 2013-11-06
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Ghodge SV;Cummings JA;Williams HJ;Raushel FM
• 通讯作者: Raushel FM

Subunit Interactions within the Carbon-Phosphorus Lyase Complex from Escherichia coli.

大肠杆菌碳磷裂解酶复合物内的亚基相互作用。

• DOI: 10.1021/acs.biochem.5b00194
• 发表时间: 2015
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Ren,Zhongjie;Ranganathan,Soumya;Zinnel,NathanaelF;Russell,WilliamK;Russell,DavidH;Raushel,FrankM
• 通讯作者: Raushel,FrankM

Potent inhibition of the C-P lyase nucleosidase PhnI by Immucillin-A triphosphate.

Immucillin-A 三磷酸有效抑制 C-P 裂合酶核苷酶 PhnI。

• DOI: 10.1021/bi4013287
• 发表时间: 2013
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Kamat,SiddheshS;Burgos,EmmanuelS;Raushel,FrankM
• 通讯作者: Raushel,FrankM

Structures of the Carbon-Phosphorus Lyase Complex Reveal the Binding Mode of the NBD-like PhnK.

• DOI: 10.1016/j.str.2015.11.009
• 发表时间: 2016-01-05
• 期刊: Structure (London, England : 1993)
• 作者: Yang K;Ren Z;Raushel FM;Zhang J
• 通讯作者: Zhang J