| Biography | |
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 Prof. Anthony J. Clarke University of Guelph, Canada |
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| Title: The Structure-Function Relationship and Inhibition of Peptidoglycan O-Acetyltransferases, Potential New Antivirulence Targets in Both Gram-Positive and Gram-Negative Bacteria | |
| Abstract: The O-acetylation of the essential bacterial cell wall polymer peptidoglycan is known to occur in a large number of bacteria including many important human pathogens, such as Staphylococcus aureus, species of Enterococcus, Helicobacter pylori, Campylobacter jejuni, and Neisseria gonnorrhoeae. This modification to the C-6 position of N-acetylmuramoyl residues of peptidoglycan inhibits the action of muramidases (lysozymes) of innate immune systems in a concentration dependant manner, and it totally precludes the activity of the lytic transglycosylases, bacterial autolysins that are involved with the insertion of flagella, pili, and secretion/transport systems, as well as the general biosynthesis and turnover of the peptidoglycan sacculus. We discovered and characterized two distinct two-component systems for the O-acetylation of peptidoglycan in Gram-positive and Gramnegative bacteria, respectively. In Gram-negative bacteria, such as N. gonorrhoeae, an integral membrane protein, peptidoglycan O-acetyltransferase (Pat) A, is proposed to translocate acetate from cytoplasmic pools of acetyl-CoA through the cytoplasmic membrane to the periplasm for its transfer to peptidoglycan by PatB. With Gram-positive bacteria, such as S. aureus, a single protein, Oacetyltransferase (Oat), appears to be a fusion of PatA and PatB to catalyze both the translocation and transfer of acetate for peptidoglycan O-acetylation. The first biochemical characterization and X-ray crystal structure of N. gonorrhoeae PatB and the C-terminal catalytic domain of OatA (OatAC) from S. pneumoniae and S. aureus are presented. The kinetic parameters for various acetyl donors and acceptors were determined for these O-acetyltransferases using a chromogenic assay coupled with MS analysis. These data indicated that both PatB and OatAC use a ping-pong, bi-bi catalytic pathway for acetyl transfer to acceptor sugars. A novel PG-based substrate was used to delineate the unique specificities for the two enzymes which account for their different temporal activities in PG metabolism. The structures of both enzymes adopt an α/β hydrolase fold comparable to SGNH esterases, and Ser-His-Asp catalytic triads were identified within active site grooves on their respective surfaces. However, a unique oxyanion loop-orientation compared to other SGNH esterases was found. Site-specific replacements confirmed the identification of these catalytic residues. The structure of OatAC was also determined in complex with a mechanism-based inhibitor covalently bound to the catalytic Ser. A mechanism of action is proposed for these O-acetyltransferases involving the formation of an acetyl-enzyme intermediate prior to the acetylation of muramoyl residues in PG. Additionally, preliminary evidence supporting the principle that these enzymes may serve as new antibiotic targets will be presented. | |
| Biography: Professor Anthony J. Clarke obtained his PhD from the University of Waterloo, Canada in Biochemistry in 1983. He joined the University of Guelph in 1986 as an assistant professor after post-doctoral stints at the Carlsberg Research Centre, Copenhagen and the National Research Council, Ottawa. Soon after moving to Guelph, he was drawn into the University's administration and he served as departmental Chair, College Dean, Assoc. V.P. (Research), Dean of Graduate Studies, and Assoc. V.P. (Graduate Studies & Program Quality Assurance). He has also served as a member of provincial and national granting agencies, associate editor of scientific journals, and he is an Editor of the Canadian Journal of Microbiology. Despite these administrative/service responsibilities, Professor Clarke has maintained an active research program that investigates the mechanism of action of carbohydrate-active enzymes to identify new targets for antibacterial therapy and to facilitate the production of cellulosic ethanol. He is the author of one book, nine book chapters, and over 110 articles in scientific journals. | |
