The Enzymology of Phosphonate Metabolism

描述(由申请人提供)：本提案中描述的研究的广泛的长期目标旨在更全面地了解酶催化反应中结构和功能之间的关系。本申请旨在提供控制蛋白质结构、底物识别和反应特异性的因素的基本分子描述。这一建议的主要重点是阐明酶催化的反应的化学机制，这些反应控制着磷酸盐到磷酸盐的代谢。磷是核酸、碳水化合物和磷脂的组成部分。有机膦化合物的代谢对人类健康具有重要意义，因为这些化合物构成了迅速增长的抗生素、除草剂、神经毒剂和抗病毒药物。然而，尽管三十多年来进行了大量的努力，但对于磷酸盐底物中失活的磷-碳键的断裂的分子描述之前还没有阐明。在原核生物中，C-P裂解酶反应的催化机制已定位于PHN基因簇。在这一应用中，C-P裂解酶复合体通过三种蛋白质：PhnI、PhnM和PhnJ的联合作用，将甲基膦酸甲酯转化为甲烷和D-核糖-1，2-环-磷酸-5-磷酸。这一应用的具体目标是确定每种酶的详细反应机理。在PhnGHL存在下，PHN I催化镁-三磷酸腺苷和甲基膦酸甲酯转化为D-核糖-1-甲基膦-5-三磷酸和腺嘌呤。然后，PhnM催化D-核糖-1-甲基膦-5-三磷酸的水解为D-核糖-1-甲基膦-5-磷酸和焦磷酸。然后，PhnJ催化D-核糖-1-甲基膦-5-磷酸转化为D-核糖-1，2-环磷酸-5-磷酸和甲烷。最终的转化需要一个[Fe4S4]-簇和S-腺苷蛋氨酸来催化活性，因此PHNJ是一种新型的自由基腺苷酸酶，它通过自由基基的中间体催化磷酸盐的P-C键的断裂。拟议的项目将为自由基-SAM类酶的机制和反应多样性提供重要的新见解，并将有助于更好地理解具有几个活性中心的多酶复合体如何能够更有效地将产物从一个活性中心引导到另一个活性中心。

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.