Janet Smith, Ph.D.

Our group studies protein structure using X-ray crystallography in order to understand protein function. We use the three-dimensional structures of proteins to understand molecular mechanisms of biological processes and to develop hypotheses about function, which we test through experiments in our lab or by collaboration. Two current projects illustrate our approach.

Glutamine amidotransferases are complex enzymes that produce ammonia from the amide group of glutamine, and channel the ammonia through an intra-molecular tunnel to a second active site where it is added to a second substrate. Amidotransferases occur in a variety of pathways for biosynthesis of nitrogenous molecules, including purine and pyrimidine bases, cofactors, aminosugars and amino acids. Our crystal structures of glutamine PRPP amidotransferase (purine pathway), GMP synthetase (guanine pathway), imidazole glycerol phosphate synthase (histidine pathway) and other enzymes show that the chemical steps of glutamine hydrolysis and amination occur independently, but that they are coordinated by the protein through a variety of conformational switches. Our analysis of the structures has shown how each enzyme keeps its glutaminase active site in an inactive state until the second substrate is bound and ready to accept the labile ammonia. Currently challenges are to understand the conformational switches used for cross-talk by the distant active sites and to study new amidotransferases that have been detected in genome sequences.

The replication machinery of alphaviruses and flaviviruses also represents a complex catalytic system. These positive-sense RNA viruses encode enzymes for genome replication, RNA capping, RNA unwinding and polyprotein processing. Specific protein-protein complexes, as yet poorly characterized, coordinate the catalytic activities and specify particular RNA or protein substrates at appropriate times in the viral life cycle. Our structural studies of individual enzymes and enzyme complexes aim to elucidate mechanisms of catalysis and of time-dependent activation and specificity.