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.
The primary objective for the research described in this proposal is directed towards an understanding of how phosphonates are converted to the essential nutrient phosphate. 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.
|Yang, Kailu; Ren, Zhongjie; Raushel, Frank M et al. (2016) Structures of the Carbon-Phosphorus Lyase Complex Reveal the Binding Mode of the NBD-like PhnK. Structure 24:37-42|
|Ren, Zhongjie; Ranganathan, Soumya; Zinnel, Nathanael F et al. (2015) Subunit Interactions within the Carbon-Phosphorus Lyase Complex from Escherichia coli. Biochemistry 54:3400-11|
|Ghodge, Swapnil V; Cummings, Jennifer A; Williams, Howard J et al. (2013) Discovery of a cyclic phosphodiesterase that catalyzes the sequential hydrolysis of both ester bonds to phosphorus. J Am Chem Soc 135:16360-3|
|Kamat, Siddhesh S; Burgos, Emmanuel S; Raushel, Frank M (2013) Potent inhibition of the C-P lyase nucleosidase PhnI by Immucillin-A triphosphate. Biochemistry 52:7366-8|