This grant proposes to collect mechanistic information on and understand the regulation of D-arabinose 5- phosphate (ASP) isomerase (API), the enzyme responsible for the synthesis of ASP from D-ribulose 5- phosphate (Ru5P), in Gram-(-) microorganisms. This information should provide validation that API, a key enzyme in lipopolysaccharide (LPS-aka endotoxin) biosynthesis, is a target for screening efforts by investigators in the field to identify selective inhibitors of API - namely a new generation of mechanistically diverse antibiotics for which no resistance is known. The goals of this project are to establish 1. the intracellular function of the ASP isomerases expressed by the genes yrbH, kpsF, c3406 and gutQ as well as to understand the regulatory role of the cystathionine beta -synthase domain (CBS) common to three of the gene products; 2. the mechanism for the formation of ASP from RuSP; and 3. the substrate specificity of the APIs and the role of active site amino acids.
The specific aims focus on diverse techniques to detect the presence of an intermediate enediol and to determine the stereochemistry of the inter-conversion of the substrate and product. Genomic knockouts will be constructed to understand the cellular function of each API as well as the potential lethality of a genomic knockout of API (target validation). Truncated derivatives of API lacking the CBS domain will be utilized to ascertain CBS domain function and a library of sugar nucleotides will be screened to find potential regulators of the CBS domain. Site-directed mutagenesis studies, based on crystallographic data and active site modification using mechanism-based irreversible inhibitors, will be exploited to gain insight into the contribution of enzyme functionalities to substrate binding, monomer interface interactions, and to the mechanism of API. The small molecules to be synthesized in this grant will serve as mechanistic probes. The ultimate goal of these studies is to better understand the role of LPS, a critical macromolecule essential to both the survival and virulence of Gram-(-) microorganisms ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI061531-02
Application #
7169212
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Korpela, Jukka K
Project Start
2006-01-15
Project End
2010-12-31
Budget Start
2007-01-01
Budget End
2007-12-31
Support Year
2
Fiscal Year
2007
Total Cost
$368,980
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Cech, David; Wang, Pan Fen; Holler, Tod P et al. (2014) Analysis of the arabinose-5-phosphate isomerase of Bacteroides fragilis provides insight into regulation of single-domain arabinose phosphate isomerases. J Bacteriol 196:2861-8
Cech, David L; Wang, Pan-Fen; Holt, Melissa C et al. (2014) A novel glucose 6-phosphate isomerase from Listeria monocytogenes. Protein J 33:447-56
Zhou, Lily; Wu, Jing; Janakiraman, Vijayalakshmi et al. (2012) Structure and characterization of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix. Bioorg Chem 40:79-86
Yep, Alejandra; Sorenson, Roderick J; Wilson, Michael R et al. (2011) Enediol mimics as inhibitors of the D-arabinose 5-phosphate isomerase (KdsD) from Francisella tularensis. Bioorg Med Chem Lett 21:2679-82
Mosberg, Joshua A; Yep, Alejandra; Meredith, Timothy C et al. (2011) A unique arabinose 5-phosphate isomerase found within a genomic island associated with the uropathogenicity of Escherichia coli CFT073. J Bacteriol 193:2981-8
Yi, Li; Velasquez, Melvin S; Holler, Tod P et al. (2011) A simple assay for 3-deoxy-d-manno-octulosonate cytidylyltransferase and its use as a pathway screen. Anal Biochem 416:152-8
Biswas, Tapan; Yi, Li; Aggarwal, Parag et al. (2009) The tail of KdsC: conformational changes control the activity of a haloacid dehalogenase superfamily phosphatase. J Biol Chem 284:30594-603
Mamat, Uwe; Meredith, Timothy C; Aggarwal, Parag et al. (2008) Single amino acid substitutions in either YhjD or MsbA confer viability to 3-deoxy-d-manno-oct-2-ulosonic acid-depleted Escherichia coli. Mol Microbiol 67:633-48
Meredith, Timothy C; Mamat, Uwe; Kaczynski, Zbigniew et al. (2007) Modification of lipopolysaccharide with colanic acid (M-antigen) repeats in Escherichia coli. J Biol Chem 282:7790-8