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）：本提案中所述研究的广泛长期目标旨在更全面地了解酶催化反应中结构与功能之间的关系。本申请旨在提供一个基本的分子描述的因素，支配蛋白质结构，底物识别，和反应特异性。该提案的主要重点是针对酶催化反应的化学机制的阐明，该酶催化反应控制膦酸盐代谢为磷酸盐。磷是核酸、碳水化合物和磷脂的组成部分。有机膦酸酯的代谢对人类健康具有重要意义，因为这些化合物构成了快速增长的一类抗生素、除草剂、神经毒剂和抗病毒药物。然而，在膦酸酯底物内的失活的磷-碳键的裂解的分子描述尚未被阐明，尽管超过三十年的努力。在原核生物中，C-P裂解酶反应的催化机制已经定位于phn基因簇。在本申请中提出，C-P裂解酶复合物通过三种蛋白质PhnI、PhnM和PhnJ的组合作用将膦酸甲酯转化为甲烷和D-核糖-1，2-环状-磷酸-5-磷酸。本申请的具体目的是确定这些酶中每一种的详细反应机制。Phn I在PhnGHL存在下催化MgATP和甲基膦酸酯转化为D-核糖-1-甲基膦酸酯-5-三磷酸和腺嘌呤。然后PhnM催化D-核糖-1-甲基膦酸酯-5-三磷酸水解为D-核糖-1-甲基膦酸酯-5-磷酸和焦磷酸。然后PhnJ催化D-核糖-1-甲基膦酸酯-5-磷酸转化为D-核糖-1，2-环状-磷酸-5-磷酸和甲烷。最终的转化需要[Fe 4S 4]-簇和S-腺苷甲硫氨酸的催化活性，因此PhnJ是一种新型的自由基SAM酶，其通过基于自由基的中间体催化膦酸酯的P-C键的裂解。该项目将为自由基SAM类酶的机制和反应多样性提供新的见解，并将有助于更好地了解具有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引导到另一个活性位点。

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