New Insights on Structure and Function of Sialyltransferases

Sialic acids are a unique posttranslational modification at the terminus of glycoproteins and -lipids. Proteins modified with oligomers of sialic acid add a repellent charge to cell surfaces, which is a crucial feature in cell migration and axonal growth during early brain development. Varied expression levels of sialic acid are linked to tumor malignancy in neuroblastoma, schizophrenia, autism and bipolar disorder but the lack thereof is linked to impaired neuronal development. On the other hand, overexpression of sialic acid oligomers in Schwann cells promotes the peripheral regeneration of lesioned nerves and improves the ability of Schwann cells to migrate into damaged tissue and to remyelinate central nervous system axons. In order to understand the molecular mechanisms of sialylation, our project focuses on the structural characterization of enzymes of the mammalian and bacterial glycosyltransferase families 29 and 42. The proteins of interest were expressed in insect cells and structural studies were undertaken by x-ray crystallography. Kinetics, SEC MALS and glycan array data will shed light on mechanism of catalysis and acceptor specificity. Altogether, the results of this study will promote further understanding of the structure-function relationship of sialyltransferases.

Analysis of bacterial cell-wall synthesis to combat antibiotic resistance

The peptidoglycan biosynthetic pathway is one of the most important processes in the bacterial cell to be exploited as a target for the design of antimicrobial drugs to combat infection and pathogenesis. This pathway, unique to bacteria, utilizes over twenty enzymes, likely in concert, with reactions that proceed from the cytoplasm, across the membrane and into the periplasmic space culminating in the production of the mesh-like structure composed of polymerized glycan and cross-linked peptide components that form the major structural component of the essential bacterial protective barrier known as the cell wall. Work in our group has aimed at understanding the structural and kinetic properties of several of these enzymes including the glycosyltransferase/transpeptidase activity of a family of enzymes known historically as the penicillin binding proteins (PBPs). As the name implies, these enzymes are also the target of beta-lactam antibiotics, and molecular modifications to transpeptidase variants have been shown to be linked to increased antibiotic resistance in superbugs such as Methicillin Resistant Staphlococcal aureus (MRSA). In parallel, highly disseminated plasmid-encoded beta-lactamase enzymes, with structural and mechanistic ties to the transpeptidases, have also arisen in many of the clinically important bacterial pathogens, leading to further widespread beta-lactam antibiotic resistance. The molecular details of these critical enzymatic reactions in bacterial viability and drug resistance will be presented.

Investigation of potential drug targets involved in antibiotic sensing and cell wall modification

The emergence of an increasing number of broad-spectrum antibiotic resistant bacteria such as MRSA over the last decade has made the understanding of the mechanisms underlying resistance critical. Resistance very often involves enzymes associated with the cell wall, a common target of antibiotics. Here we investigate the structure and function of enzymes involved in antibiotic sensing and cell wall modification, while using a customized "cell-free" protein synthesis approach to obtain sufficient yields of promising but difficult-to-express antibacterial drug targets.