A major health problem today is an increasing resistance in pathogenic bacteria to the classic antibacterials, such as the penicillins. A potential 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. Many pharmacologically important drugs act by inhibiting key enzymes in various primary and secondary biochemical cascades. To be successful in this approach one needs a thorough understanding of the enzyme(s) at the molecular level. The goal of our group is to use information obtained from substrate analogue studies, x-ray crystallographic structure determination in parallel with studies utilizing regio- and stereospecific labelled substrates and information from kinetics methodologies, in the design and synthesis of selective enzyme inhibitors of pivotal enzymes in critical biosynthetic cascades.
PEP analogues and various carbohydrate derivatives are being prepared to investigate the biosynthesis of the pharmacologically important 3-deoxy-2-keto acid sugars 3-deoxy-D-arabino-2-heptulosonic acid phosphate (DAH7P) (a key intermediate in the microbial biosynthesis of the aromatic amino acids phenylalanine, tyrosine and tryptophan as well as other aromatic compounds) and 3-deoxy-D-manno-2-octulosonic acid phosphate (KDO8P) (a required acidic sugar in the biosynthesis of the lipid A portion of the LPS of G- microorganisms). Ribulose 5-phosphate isomerase and KDO8P phosphatase as well as a key enzyme in the biosynthesis of intermediate in the biosynthesis of the cell wall component peptidoglycan, UDP-NAcGlc-enol pyruvate (the branch point between nucleotide-sugar metabolism and N-acetylmuramyl peptide synthesis) are under study. The enzymes responsible for the biosynthesis of each of these carbohydrates represent important chemotherapeutic targets for the design of more selective drug molecules.
Other labeled probes and substrate analogues are being used to study the mechanism of the enzyme-catalyzed biosynthesis of the unusual amino acids 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor to the important plant hormone, ethylene and L-azetidine-2-carboxylic acid, a naturally-occurring toxic proline analogue.
The common experimental aspects of each of these studies is synthesis, some involving isotopic labeling techniques, spectroscopic analysis (mainly NMR), manipulation of microorganisms, cloning and overexpression of target enzymes, genetic manipulation including site-directed mutagenesis, enyzme isolation and purification and enzyme kinetics.
Wu, Jing and Ronald W. Woodard, Journal of Biological Chemistry , 278 , 18117-18123 (2003) "YrbI is the Specific 3-Deoxy-D- manno -Octulosonate 8-Phosphate Phosphatase in Escherichia coli."
Howe, David L., Appavau K. Sundaram, Domenico L. Gatti and Ronald W. Woodard, Biochemistry , 42 , 4843-4845 (2003) "Mechanistic Insight into 3-deoxy-D- manno -octulosonate-8-phosphate Synthase ( KDO8-P Synthase) and 3-deoxy-D- arabino- heptulosonate-7-phosphate Synthase (DAH7-P Synthase) Utilizing Phosphorylated Monosaccharide Analogues."
Wu, Jing, David L. Howe, and Ronald W. Woodard, Journal of Biological Chemistry , 278 , 27525-27531 (2003) " Thermotoga maritima 3-Deoxy- d - arabino -heptulosonate 7-phosphate Synthase: The Ancestral Eubacterial DAHP Synthase?"
Meredith, Tim and Ronald W. Woodard, Journal of Biological Chemistry, 278 , 32771-32777 (2003) " Escherichia coli YrbH is a D-Arabinose 5-phosphate Isomerase ."
Li, Jingjing , Jing Wu, Angela S. Fleischhacker, and Ronald W. Woodard , Journal Of American Chemical Society , 126 , 7448-7449 (2004) "Conversion of Aquifex a eolicus 3- D eoxy- d - manno -octulosonate 8-phosphate Synthase, a Metalloenzyme, into a Non-metalloenzyme. "
Shumilin, Igor A., Ronald Bauerle, Jing Wu, Ronald W. Woodard, and Robert H. Kretsinger, Journal of Molecular Biology, 341 , 455-466 (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."