Microbes and host organisms continuously interact with each other and haveco-evolved delicate systems to cope with these interactions for survival. Investigating thisimportant relationship will lead to better understanding of the pathogenesis mechanism ofpathogenic bacteria for better protection as well as better utilization of the symbioticinteraction that will enhance the control of biomass accumulation in agriculture. Structuralbiology is an indispensible approach that can provide us information at the molecular levelto better understand the mechanism of the microbe-host interaction and to develop bettertreatments. In this thesis, three important proteins, namely YpfP from Bacillus subtilis,Sus1 from Arabidopsis thaliana and Eha proteins from pathogenic Escherichia coli O157:H7 were investigated by biochemical characterization as well as X-ray crystallography toprovide the knowledge base for future research.Structures of AtSus1 in complex with UDP and fructose were determined at resolutionsof 2.8 Å and 2.85 Å, respectively. These structures provided insights into sucrose synthesisand cleavage as well as the potential link between the AtSus1 activity and its spatialinteractions with cellular targets. They also helped to elucidate the retainingglycosyltransferase mechanism in a broader aspect. In addition, comparison of the retainingglycosyltransferase AtSus1 with an inverting glycosyltransferase revealed an interestingrelationship that the substrate specificity between the two might be related by a simpledomain rotation.Structures of EhaA, B, and D proteins from pathogenic E. coli O157: H7 werestudied by X-ray crystallography. Diffraction data were collected for EhaA, B, and DC-terminal β domains with the structure of EhaB_c successfully solved to 2.2 Å. Crystalswere also obtained for EhaA and EhaB full length proteins but further pursuit of crystalswith better diffraction quality are still required. Analysis of the EhaB_c structure revealedan interesting difference with other solved structures of the autotransporter translocationunits: EhaB_c has a significantly higher number of bulky aromatic residues inside thebarrel and a narrower channel indicated by the lower conductance measured in planarBilayer Lipid Membranes experiments. However, how these preliminary observations arerelated to the adhesion function of the Eha proteins is still unknown. Further detailedanalysis of structures and activity of Eha proteins is still needed to elucidate thisstructure-function relationship.A giant liposome assay was developed to assay the glycosyl transfer activity ofBsYpfP. It transfered two glucose moieties to the diacylglycerol with lipid chain varyingfrom C10:0 to C18:1 in this assay. The initial unsuccessful efforts of crystallizing BsYpfPled to an investigation of the fusion module method to facilitate its crystallization. Crystalswere obtained with the N-terminal fusion protein of the helix-turn-helix module from TagFprotein, but they diffracted poorly. However, this effort offered a clue for the crystallizationconditions as well as valuable experience on this method. Further manipulation andcharacterization are still needed to obtain diffraction quality crystals.
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