Structural Characterization Of Enzymes Involved In Biosynthesis Of Thiamin And Salvage Of Uridine

Abstract

Macromolecular X-ray crystallography was used for structural characterization of enzymes involved in thiamin biosynthetic and uridine salvage pathways. The enzymes involved in major metabolic pathways are potential drug targets and understanding their structural features and active site details lead to development of effective inhibitors. THI6 is a bifunctional enzyme present in the eukaryotic thiamin biosynthetic pathway. The N-terminal domain of this enzyme catalyses the ligation of the thiamin thiazole and pyrimidine moieties to form thiamin phosphate and the Cterminal domain catalyzes the phosphorylation of 4-methyl-5-(hydroxyethyl)thiazole in a salvage pathway. In lower organisms, thiamin phosphate synthase and 4-methyl5-(hydroxyethyl)thiazole kinase are separate gene products. Current work reports the first crystal structure of a eukaryotic THI6 along with several complexes that characterize the active sites responsible for the two chemical reactions. THI6 from Candida glabrata is a homehexamer in which the six protomers form a cage like structure. Two domains within a protomer interact via two loop regions not found in the bacterial enzymes. The structures of different protein-ligand complexes define the active sites of the two domains. Our structural studies reveal that the active sites of the two domains are 40 Amgstrums apart and are not connected by an obvious channel. The present work also includes structural and biochemical characterization of THI1 from Zea maize, a thiazole synthase involved in eukaryotic thiamin biosynthetic pathway. The structural work was performed to investigate the role of the residue Val211 in this particular enzyme, as genetic mapping showed that Val211Met mutation resulted in a phenotype called Bladekiller 1. In this phenotype plants develop some unusual characteristics, such as poor development of leaf blades, premature termination of meristems etc. The three dimensional structure of the wildtype enzyme in a complex form with the product have been described. Biochemical assays reveal that the wild type enzyme purified with the bound metabolites but the mutant does not. The dynamic light scattering studies show that the wild type enzyme forms an octamer in solution state unlike the mutant enzyme. Finally the three dimensional structure of uridine phosphorylase, a key enzyme in the pyrimidine salvage pathway was described with the formation of glycal in its active site. This enzyme catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate (or 2'-deoxyuridine to 2'-deoxyribose 1-phosphate). The reaction is believed to proceed via an oxocarbenium ion-like transition state. The reported structures include Escherichia coli uridine phosphorylase treated with 5fluorouridine and sulfate and dimeric bovine uridine phosphorylase treated with 5fluoro-2'-deoxyuridine or uridine, plus sulfate. In each case the electron density shows three separate species corresponding to the pyrimidine base, sulfate and a ribosyl species. NMR time course studies demonstrated that uridine phosphorylase can catalyze the hydrolysis of these fluorinated nucleosides in the absence of phosphate or sulfate. Crystallization with the hydrolysis products in the presence of sulfate is then proposed to result in the anti-elimination of water across the C1-C2 bond of UPasebound ribose. In the structures of the glycal complexes the fluorouracil O2 atom is appropriately positioned to act as the general base required for this elimination reaction. Crystals of bovine uridine phosphorylase treated with 2'-deoxyuridine and sulfate show intact nucleoside. This nucleoside is unactivated toward cleavage of the N-ribosyl bond in the absence of phosphate. These results add a previouslyunencountered mechanistic motif to the body of information on glycal formation catalyzed by enzymes which catalyze the cleavage of glycosyl bonds.