STRUCTURAL STUDIES OF METABOLIC ENZYMES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our laboratory studies the structures and functions of metabolic enzymes. We are interested in the catalytic mechanisms of individual enzymes, protein:protein interactions between enzymes within a pathway, evolution of protein function, and drug design. PurP is an enzyme in the basic pathway for the biosynthesis of inosine 5`-monophosphate (IMP). Purine nucleotides are the building blocks of RNA and DNA and enzymes of the purine biosynthetic pathway are targets for anticancer, antiviral and antimicrobial drug discovery. The crystals diffract to reasonably high resolution and soaks will help better characterize the active site and provide missing structural information. In Archaea, genes responsible for the first two steps of the aromatic amino acid biosynthesis leading to 3-dehydroquinate (DHQ) are missing. Recently, two protein products from Methanocaldococcus jannaschii, Mj0400 and Mj1249, have been reported as the nonorthologous catalysts of the DHQ formation. The crystal structures of Mj400 will further assist the identification of the substrates, and the elucidation of their catalytic mechanisms. Crystals have been soaked in effort to bind small molecules at the active site to help provide this information. Uridine Phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway. It catalyses the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. The inhibition of UP in human increases blood uridine levels and produces a protective effect against the toxicity of the chemotherapeutic agent 5-fluorouracil without reducing its antitumor activity. Present studies are focused on structural characterization and mechanistic understanding of bovine uridine phosphorylase and the design of new inhibitors. New soaks will help characterize the active site. The lab has published seven papers on enzymes in the purine biosynthetic pathway and two papers on uridine phosphosphorylase from other organisms. Aromatic amino acid biosynthesis is a new area of interest for the group.