AdvisorSundberg, Eric J.
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AbstractEnzymes are biological catalysts that enable life by accelerating specific reactions. As new infectious diseases are discovered, the enzymes produced by these organisms require characterization. In some cases, they can be inhibited to prevent disease; in other cases, they can be coopted or altered to serve as diagnostic or therapeutic tools. In this work, we explore the mechanisms of several enzymes involved in infection and immunity. In the first major theme, we characterize the mechanisms of fosfomycin resistance proteins from Escherichia coli (FosA3) and Klebsiella pneumoniae (FosAKP), which degrade the antibiotic fosfomycin. Although the active sites of FosA enzymes are well conserved, differences in activity between enzymes may be due to the presence of an allosteric site at the dimer interface of these enzymes. We solved crystal structures with a novel small molecule FosA inhibitor, termed ANY1, which binds with high affinity to the active site and acts as a competitive inhibitor of fosfomycin binding. By inhibiting FosA, ANY1 potentiates the effects of fosfomycin in several priority Gram-negative pathogens, and may serve as a suitable lead candidate for structure-guided drug design and pre-clinical development. In the second major theme, we describe the mechanisms of enzymes with activity on the carbohydrates of IgG antibodies. EndoS and EndoS2 are enzymes produced by Streptococcus pyogenes, which remove the conserved glycan on Asn297 of IgG to evade the host immune system. We discovered that these enzymes share a conserved mechanism of substrate recognition, binding primarily to the core and α(1,3) antenna of the IgG glycan. EndoS2 recognizes a more diverse set of glycans through differences in the both the active site and a coevolved carbohydrate binding module. We then describe the structure and function of AlfC, an α(1,6)-specific fucosidase from Lactobacillus casei with activity on the core fucose of antibodies. We identified D200 and D242 as the most likely catalytic residues, and provide a structural basis for the remarkable specificity of this enzyme and transfucosidase mutants. Finally, we describe a novel application for these carbohydrate-active enzymes in the directed evolution of antibodies based on yeast display.
Molecular Microbiology and Immunology
University of Maryland, Baltimore