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Inhibition Studies of DapL Enzymes Orthologs and Synthesis of Proposed Biosynthetic Intermediates of Tabtoxin and Synthesis of a Hexaketide as a Standard for Lovastatin Studies Open Access


Other title
Type of item
Degree grantor
University of Alberta
Author or creator
Rodriguez-Lopez, Eva M
Supervisor and department
Vederas, John (Department of Chemistry)
Examining committee member and department
Gänzle, Michael (Department of Agricultural, Food & Nutritional Science
Li, Liang (Department of Chemistry)
Clive, Derrick (Department of Chemistry)
Auclair, Karine (McGill University)
Cairo, Christopher (Department of Chemistry)
Department of Chemistry

Date accepted
Graduation date
2017-11:Fall 2017
Doctor of Philosophy
Degree level
The enzyme LL-diaminopimelate aminotransferase (DapL) catalyzes the transformation of L-THDP to LL-DAP during the biosynthesis of lysine in plants, alga, and in some pathogenic bacteria like Leptospira interrogans and Verrucomicrobium spinosum. Because the biosynthesis of lysine is absent in mammals, specific inhibitors of DapL enzyme could potentially function as antibiotics with low toxicity to humans or as herbicides or algaecides. An inhibition study of DapL enzyme from three orthologs (V. spinosum, L. interrogans, and C. reinhardtii) was completed. Five potential pharmacophores were selected (derivatives of rhodanine, barbiturate, and thiobarbiturate). The results showed a different inhibition pattern between each ortholog. In order to help to understand the differences we used structural modeling and protein alignment of DapL orthologs. Tabtoxin is a phytotoxic dipeptide from P. syringae that contains a non-proteinogenic amino acid, tabtoxinine-β-lactam (TβL). Once the peptide bond in tabtoxin is hydrolysed by aminopeptidases in the host or in the periplasm of the pathogen, TβL is released. TβL is the actual toxin because it is responsible for irreversible inactivation of glutamine synthetase. For example, this inhibition results in accumulation of high levels of ammonia causing chlorosis on the leaves, in tobacco wildfire disease. To date, the intermediates involved in the biosynthesis of tabtoxin, have not been elucidated. In this work we proposed a complete biosynthesis of tabtoxin. In order to demonstrate this pathway, synthesis of three proposed intermediates in the biosynthesis of tabtoxin were pursued. Synthetic methodologies were developed towards the synthesis of labeled and/or unlabeled intermediates. Lovastatin is a statin that reduces cholesterol levels through the inhibition of the enzyme HMG-CoA reductase. The biosynthesis of lovastatin is accomplished by a HR-PKS, LovB with involvement of LovC in Aspergillus terreus. LovB has a condensation (CON) domain of a non-ribosomal peptide synthetase (NRPS). CON domain is believed to be a natural Diels-Alderase that catalyzes the formation of the decalin ring. In this project the synthesis of a N-acetyl cysteamine (SNAC) ester of a hexaketide was pursued. This hexaketide may be used as a standard for GC-MS to investigate the substrate specificity of LovB. The enzymatic study would demonstrate if the CON domain is responsible for catalyzing the Diels-Alder reaction during the biosynthesis of lovastatin.
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
Citation for previous publication
McKinnie, S. M. K.; Rodriguez-Lopez, E. M.; Vederas, J. C.; Crowther, J. M.; Suzuki, H.; Dobson, R. C. J.; Leustek, T.; Triassi, A. J.; Wheatley, M. S.; Hudson, A. O., Differential response of orthologous L,L-diaminopimelate aminotransferases (DapL) to enzyme inhibitory antibiotic lead compounds. Bioorganic & Medicinal Chemistry 2014, 22 (1), 523-530. DOI: 10.1016/j.bmc.2013.10.055.Cochrane, S. A.; Findlay, B.; Bakhtiary, A.; Acedo, J. Z.; Rodriguez-Lopez, E. M.; Mercier, P.; Vederas, J. C., Antimicrobial lipopeptide tridecaptin A1 selectively binds to Gram-negative lipid II. Proceedings of the National Academy of Sciences 2016, 113 (41), 11561-11566. DOI: 10.1073/pnas.1608623113.

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