Structural and functional studies of folylpolyglutamate synthetase.

Description

Folylpolyglutamate synthetase (FPGS) catalyzes the polyglutamylation of folates and antifolates. FPGS is an essential enzyme, required in bacteria for folate biosynthesis and in mammals for folate retention in cells and in organelles, and itself is also a potential drug target for cancer chemotherapy and treatment of microbial infection. I have undertaken structural and functional studies of FPGS with the goal of gaining better understanding of the mechanism of FPGS.Using Electronic Paramagnetic Resonance (EPR) and the site-directed spin labeling (SDSL) technique, I detected conformational changes of FPGS upon binding ATP or folate. This study not only complements the crystal structure data but also provided dynamic information on FPGS in the solution state for the first time.I have characterized the conserved residues at the active site of Lactobacillus casei (L. casei) FPGS, using site-directed mutagenesis, enzyme assays and kinetic experiments. This study provided biochemical information for the folate- and glutamate-binding sites of FPGS and determined the functional roles for each mutated residue.The difference between the L. casei FPGS and E. coli enzyme lies in the fact that the former has only FPGS activity and the latter functions as both FPGS and dihydrofolate synthetase. I have made chimeric proteins of L. casei and E. coli FPGS and characterized them using enzyme specific activity assay, equilibrium dialysis and fluorescence. This study showed that there is no additional domain required for DHFS activity. In addition, it provided information on the structural determinants for folate substrate specificity.I have characterized an essential glutamate residue of FPGS (Glu143 in L. casei FPGS), which has been proposed to coordinate the Mg 2+ ion in the crystal structure of L. casei FPGS. Using site-directed mutagenesis, equilibrium dialysis and Thin-layer chromatography (TLC) assay, I was able to show that the glutamate mutant had increased ATPase activity and was defective in transferring the gamma-phosphate of ATP to folate. This result not only demonstrated the importance of Glu143 in FPGS function, but also shed light on the working mechanism of enzyme catalysis.

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