2026-09-19T19:10:34Zhttps://dspace.mit.edu/server/oai/requestoai:dspace.mit.edu:1721.1/874712026-06-17T14:45:42Zcom_1721.1_7582com_1721.1_7581col_1721.1_131022
Barbara Imperiali.
Morrison, Michael James
Massachusetts Institute of Technology. Department of Chemistry.
Massachusetts Institute of Technology. Department of Chemistry
2014-05-23T19:34:55Z
2014-05-23T19:34:55Z
2013
2014
http://hdl.handle.net/1721.1/87471
879662515
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, February 2014.
Cataloged from PDF version of thesis. "February 2014."
Includes bibliographical references.
The highly-modified, bacterial sugar N,N'-diacetylbacillosamine (diNAcBac) has been implicated in the pathogenicity of certain microbes through its incorporation onto various protein virulence factors. In particular, diNAcBac is found at the reducing end of glycans in both asparagine (N-linked) and serine/threonine (0-linked) protein glycosylation pathways. The second and third chapters examine the O-linked protein glycosylation pathway responsible for the biosynthesis of the UDP-diNAcBac nucleotide sugar in Neisseria gonorrhoeae and Acinetobacter baumannii. UDP-diNAcBac is biosynthesized from UDP-N-acetylglucosamine through the action of a dehydratase, aminotransferase, and acetyltransferase. Specifically, these enzymes are purified, biochemically characterized, and compared to the N-linked pathway proteins from Campylobacterjejuni. Furthermore, the substrate specificity of the A. baumannii phosphoglycosyltransferase that catalyzes the transfer of UDP-diNAcBac onto undecaprenylphosphate is determined. The fourth chapter explores the structural characterization of the acetyltransferases from the O-linked protein glycosylation pathways in N. gonorrhoeae (PglB-ATD) and A. baumannii (Weel). These enzymes are members of the left-handed [beta]-helix family and are responsible for the acetylation of UDP-2-acetamido-4-amino-2,4,6-trideoxy-[alpha]-D-glucose (UDP-4-amino) to produce UDP-diNAcBac. Based upon these structures, a series of active site mutations are generated and kinetically characterized for both the AcCoA and UDP-4-amino substrates. These results suggest that although each enzyme catalyzes the acetyltransferase reaction with identical substrates, key residues within the binding pockets can lead to a diverse set of catalytic efficiencies. The final three chapters investigate the inhibition of UDP-diNAcBac pathway enzymes in C. jejuni, N. gonorrhoeae, and A. baumannii. The fifth chapter explores a fragment-based approach to identify small molecules that inhibit the aminotransferase in C. jejuni. To this end, a crystal structure of this protein is solved in complex with a fragment molecule and analogs of this compound synthesized. The sixth chapter identifies small molecule acetyltransferase inhibitors through a high-throughput screening effort in collaboration with the Broad Institute. Lastly, the seventh chapter describes a fragment-based approach to establish small molecule inhibitors for the acetyltransferase from N gonorrhoeae.
by Michael James Morrison.
Ph.D.
262 pages
eng
Massachusetts Institute of Technology
M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.
http://dspace.mit.edu/handle/1721.1/7582
Chemistry.
Elucidation of the pathways responsible for the biosynthesis of UDP-N,N'-diacetylbacillosamine in bacterial pathogens
Thesis
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Publication
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<Abstract>The highly-modified, bacterial sugar N,N'-diacetylbacillosamine (diNAcBac) has been implicated in the pathogenicity of certain microbes through its incorporation onto various protein virulence factors. In particular, diNAcBac is found at the reducing end of glycans in both asparagine (N-linked) and serine/threonine (0-linked) protein glycosylation pathways. The second and third chapters examine the O-linked protein glycosylation pathway responsible for the biosynthesis of the UDP-diNAcBac nucleotide sugar in Neisseria gonorrhoeae and Acinetobacter baumannii. UDP-diNAcBac is biosynthesized from UDP-N-acetylglucosamine through the action of a dehydratase, aminotransferase, and acetyltransferase. Specifically, these enzymes are purified, biochemically characterized, and compared to the N-linked pathway proteins from Campylobacterjejuni. Furthermore, the substrate specificity of the A. baumannii phosphoglycosyltransferase that catalyzes the transfer of UDP-diNAcBac onto undecaprenylphosphate is determined. The fourth chapter explores the structural characterization of the acetyltransferases from the O-linked protein glycosylation pathways in N. gonorrhoeae (PglB-ATD) and A. baumannii (Weel). These enzymes are members of the left-handed [beta]-helix family and are responsible for the acetylation of UDP-2-acetamido-4-amino-2,4,6-trideoxy-[alpha]-D-glucose (UDP-4-amino) to produce UDP-diNAcBac. Based upon these structures, a series of active site mutations are generated and kinetically characterized for both the AcCoA and UDP-4-amino substrates. These results suggest that although each enzyme catalyzes the acetyltransferase reaction with identical substrates, key residues within the binding pockets can lead to a diverse set of catalytic efficiencies. The final three chapters investigate the inhibition of UDP-diNAcBac pathway enzymes in C. jejuni, N. gonorrhoeae, and A. baumannii. The fifth chapter explores a fragment-based approach to identify small molecules that inhibit the aminotransferase in C. jejuni. To this end, a crystal structure of this protein is solved in complex with a fragment molecule and analogs of this compound synthesized. The sixth chapter identifies small molecule acetyltransferase inhibitors through a high-throughput screening effort in collaboration with the Broad Institute. Lastly, the seventh chapter describes a fragment-based approach to establish small molecule inhibitors for the acetyltransferase from N gonorrhoeae.</Abstract>
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