A problem confronting present day health care providers is an increasing resistance in pathogenic bacteria to the classic antibacterials. An approach to overcoming this problem is to design new and innovative agents with a totally different mode of action; therefore, no cross-resistance with present therapeuticals should occur. Most antimicrobial drugs act by inhibiting key enzymes in the biosynthesis of macromolecular molecules necessary for viability of the microorganism. Success in this type of approach necessitates a thorough understanding of the enzyme(s) at the molecular level. The goal of this work is to collect mechanistic information about the enzyme 3-deoxy-D-manno-octulosonic 8-phosphate synthase. The information collected in these investigations should prove useful in the future design and synthesis of selective enzyme inhibitors of this unique enzyme, namely a new generation of mechanistically diverse gram negative antibiotics.
The specific aim of this project is to establish the mechanism for the formation of 3-deoxy-D-manno-octulosonic 8-phosphate (KDO 8-P) from arabinose 5-phosphate (A 5-P) and phosphoenol pyruvate (PEP) catalyzed by the enzyme KDO 8-P synthase (EC 4.1.2.16) (KDO 8-P S), the enzyme which catalyzes one of the first committed steps in the biosynthesis of the lipid A portion of the lipopolysaccharide region of the cell envelope of gram-negative bacteria. Steady-state and stopped-flow kinetics will be used to determine the order of substrate(s) binding and product release as well as provide some insight into the potential intermediate(s) for NMR structural studies; multinuclear NMR analysis of the interaction of KDO 8-P synthase with regio- and stereo-specific labeled substrate and product analogs, several designed to """"""""visualize"""""""" the potential transient intermediate(s), and active site modification employing substrate(s)/product protection, coupled with site-directed mutagenesis studies will be exploited to gain further insight into the contribution of various enzyme functionalities to substrate binding and the mechanism of the enzyme, in particular active site cysteines. An X-ray crystallographic study will provide information concerning the three-dimensional structure of the enzyme in the solid state. The experimental strategies are designed to allow probing of several mechanistic possibilities.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053069-04
Application #
6019080
Study Section
Special Emphasis Panel (ZRG3-BNP (02))
Project Start
1996-07-01
Project End
2001-03-31
Budget Start
1999-07-01
Budget End
2001-03-31
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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
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
Wu, Jing; Woodard, Ronald W (2006) New insights into the evolutionary links relating to the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase subfamilies. J Biol Chem 281:4042-8
Wu, Jing; Sheflyan, Galina Ya; Woodard, Ronald W (2005) Bacillus subtilis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase revisited: resolution of two long-standing enigmas. Biochem J 390:583-90
Sundaram, Appavu K; Pitts, Lee; Muhammad, Kamilah et al. (2004) Characterization of N-acetylneuraminic acid synthase isoenzyme 1 from Campylobacter jejuni. Biochem J 383:83-9
Li, Jingjing; Wu, Jing; Fleischhacker, Angela S et al. (2004) Conversion of aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase, a metalloenzyme, into a nonmetalloenzyme. J Am Chem Soc 126:7448-9
Furdui, Cristina; Zhou, Lily; Woodard, Ronald W et al. (2004) Insights into the mechanism of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (Phe) from Escherichia coli using a transient kinetic analysis. J Biol Chem 279:45618-25
Woodard, Ronald W (2004) Unique biosynthesis of dehydroquinic acid? Bioorg Chem 32:309-15
Wu, Jing; Patel, Mayur A; Sundaram, Appavu K et al. (2004) Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase. Biochem J 381:185-93
Shumilin, Igor A; Bauerle, Ronald; Wu, Jing et al. (2004) Crystal structure of the reaction complex of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Thermotoga maritima refines the catalytic mechanism and indicates a new mechanism of allosteric regulation. J Mol Biol 341:455-66

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