NSF Award
0235122
The goal of this project is to gain insight into the pivotal role that phosphotransfer reactions play in biology, and to explore the range of activities performed by proteins in a structural family. In addition to proteins whose function are already known, the enzyme family members include proteins emerging from genome sequencing projects, that the structural/sequence information available so far indicates that they are incorrectly annotated in sequence databases. The information gained will be used to relate active site structure to catalysis, and to identify markers, which can be applied in the assignment of function to other proteins from each family studied. These studies will lead to the discovery of novel biochemistry and pathways, and will provide insights into the evolution of function from ancestral active site templates. Two proteins provide the framework for selection of sequence families: (1) Pyruvate phosphate dikinase (PPDK), a multi-domain enzyme that catalyzes the inter-conversion of ATP, Pi and pyruvate with AMP, PPi and phosphoenolpyruvate (PEP). (2) PEP mutase, an enzyme that catalyzes the rearrangement of PEP to phosphonopyruvate, a reaction that serves as the major entry step into the synthesis of all natural phosphonates. This is a structure/function collaborative effort, focused on structural data that are obtained by x-ray crystallography. The x-ray structures provide the basis for understanding enzyme mechanisms, and for testing proposals by site-directed mutagenesis and inhibitor design. In addition to the above proteins, new proteins that will be investigated include (1) a PPDK homologue from M. tuberculosis, Rv1127c, lacking the pyruvate binding-domain, and therefore expected to lead to the discovery of a novel phosphotransfer pathway; (2) members of the PEP mutase/isocitrate lyase enzyme family, with special emphasis on a protein associated with carnation flower petal death, which is annotated as a mutase in sequence databases, but is actually a novel lyase.<br/><br/>Broader Impact: The project will provide research opportunities to students at all levels: post-doctoral fellows, graduate students, and undergraduate students. It will lead to the discovery of novel protein functions and novel biological pathways. The results will provide new targets for biotechnological exploitation, and will be disseminated by publication in scientific journals, by depositing the coordinates in the Protein Data Bank, and by correcting annotations in sequence databases.
