Galactofuranose (Galf) residues have been implicated in the virulence or viability of many microbes, including mycobacteria. The goal of the proposed research is to understand the mechanisms underlying Galf residue incorporation into the mycobacterial cell wall. We shall investigate the structure, catalytic mechanism, and function of two key enzymes in this process: the flavoenzyme uridine-5'-diphosphate (UDP)-galactopyranose mutase (Glf or UGM) and the galactosylfuranosyltransferase GlfT2. The three Specific Aims of this application follow.
Aim 1 is to understand the mechanism of the flavoenzyme UGM. Elucidating the catalytic mechanism UGM will enhance our understanding of the diverse chemistry of the flavoenzymes, provide insight into the chemistry underlying cell wall biosynthesis, and guide the generation of inhibitors of this essential enzyme.
Aim 2 is to generate potent and cell-permeable inhibitors of UGM that can be used as probes of cell wall biosynthesis and as leads for the development of new antimycobacterial agents.
Aim 3 is to investigate the enzyme GlfT2, which catalyzes the synthesis of a galactan polymer composed of alternating 1,5- and 1,6-linked Galf residues. We shall test whether the polymerization is processive, explore how a single enzyme generates two regioisomeric sugar linkages, and determine how polymer length is controlled. These investigations will illuminate the mechanisms underlying galactan biosynthesis in mycobacteria and the biosynthesis of polysaccharides, in general. In pursuing these Aims, we shall employ methods and ideas from organic chemistry, glycobiology, carbohydrate chemistry, chemical enzymology, structural biology, microbiology, and chemical biology. Significance: The results of the proposed research will provide new insights into the assembly of the galactan polymer, an essential component of the mycobacterial cell wall. They also will address the fundamental question of how biological systems control polymer length in the absence of a template. This knowledge will be applied to develop small molecules that block mycobacterial cell growth. Such agents will serve as valuable probes of mycobacterial cell wall biosynthesis and as leads for the development of new antimycobacterial drugs.

Public Health Relevance

This research project is focused on understanding essential steps in the biosynthesis of the mycobacterial cell wall. Mycobacteria cause a number of diseases, including tuberculosis (TB). TB causes about 1.7 million deaths each year, and current therapies are failing. The goal of this project is to understand key steps in mycobacterial cell wall biosynthesis that are not targeted by any current drugs and find inhibitors from which new types of drugs could be developed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI063596-09
Application #
8602785
Study Section
Special Emphasis Panel (ZRG1-BCMB-U (02))
Program Officer
Boyce, Jim P
Project Start
2005-03-15
Project End
2014-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
9
Fiscal Year
2014
Total Cost
$320,413
Indirect Cost
$97,663
Name
University of Wisconsin Madison
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Martinez Farias, Mario A; Kincaid, Virginia A; Annamalai, Venkatachalam R et al. (2014) Isoprenoid phosphonophosphates as glycosyltransferase acceptor substrates. J Am Chem Soc 136:8492-5
Kraft, Matthew B; Martinez Farias, Mario A; Kiessling, Laura L (2013) Synthesis of lipid-linked arabinofuranose donors for glycosyltransferases. J Org Chem 78:2128-33
Wesener, Darryl A; May, John F; Huffman, Elizabeth M et al. (2013) UDP-galactopyranose mutase in nematodes. Biochemistry 52:4391-8
May, John F; Levengood, Matthew R; Splain, Rebecca A et al. (2012) A processive carbohydrate polymerase that mediates bifunctional catalysis using a single active site. Biochemistry 51:1148-59
Brown, Christopher D; Rusek, Max S; Kiessling, Laura L (2012) Fluorosugar chain termination agents as probes of the sequence specificity of a carbohydrate polymerase. J Am Chem Soc 134:6552-5
Levengood, Matthew R; Splain, Rebecca A; Kiessling, Laura L (2011) Monitoring processivity and length control of a carbohydrate polymerase. J Am Chem Soc 133:12758-66
Dykhuizen, Emily C; Kiessling, Laura L (2009) Potent ligands for prokaryotic UDP-galactopyranose mutase that exploit an enzyme subsite. Org Lett 11:193-6
Gruber, Todd D; Westler, William M; Kiessling, Laura L et al. (2009) X-ray crystallography reveals a reduced substrate complex of UDP-galactopyranose mutase poised for covalent catalysis by flavin. Biochemistry 48:9171-3
May, John F; Splain, Rebecca A; Brotschi, Christine et al. (2009) A tethering mechanism for length control in a processive carbohydrate polymerization. Proc Natl Acad Sci U S A 106:11851-6
Dykhuizen, Emily C; May, John F; Tongpenyai, Aimon et al. (2008) Inhibitors of UDP-galactopyranose mutase thwart mycobacterial growth. J Am Chem Soc 130:6706-7