The success of several sequence genomics projects heralds a new era in the study of the gene products and many of them are attractive targets for drug design. The new proteomics research uses multidisciplinary approach to study the """"""""cellular protein universe"""""""" using several high-throughput methods. X-ray crystallography plays a pivotal role in both proteomics and structure based drug design. The University of Kansas Medical Center has envisioned a plan to establish """"""""Kansas City Proteomics Initiative"""""""" and will set up an X-ray crystallography lab as a part of this plan in the Department of Biochemistry. This will serve as the Core facility for COBRE investigators who apply crystallography to their research. Funds are requested from COBRE for its maintenance. The Principal Investigator will direct the Core and his Research Project is summarized below. Gram-negative bacteria Neisseria meningitidis and Escherichia coli K1 are associated with sepsis and meningitis. There is no promising vaccine against N. Meningitidis group B and E. coli K1. Their capsular polysaccharides are sialylated, which are structurally similar to those found in mammalian glycolipids. This host mimicry plays and important role in the pathogenecity by allowing the bacteria to evade the host's immune system. Thus, sialic acid (N-acetylneuraminic acid, NeuNAc) is a virulence factor in these pathogenic bacteria. The biosynthesis of polysialic acid involves the formation and activation of NeuNAc. In bacteria, these two steps are catalyzed by NeuNAc synthase and CMP-NeuNAc synthetase, respectively. Our long-term goal is to study the structure-function relations of these enzymes using molecular biology, protein chemistry, and crystallography methods and use the structural knowledge of rational drug design.
The specific aims of this application are:
Aim 1 : NeuNAc synthases from N. meningitidis and E. coli K1. In bacteria, sialic acid is synthesized by the condensation of N-acetylmannosamine with polyenolpyruvate by NeuNAc synthase. The human enzyme uses the phosphorylated substrates. The differences in the substrate specificity between human and bacterial enzymes may be due to different active site structures. Selective inhibition of bacterial enzyme is a viable strategy to prevent the synthesis of sialic acid in the pathogen. We will overexpress, purify, and crystallize NeuNAc synthases from N. meningitidis and E. coli K1 for structural-functions studies.
Aim 2 : CMP-NeuNAc synthetase from E. coli K1. The E coli K1 CMP-NeuNAc synthetase is twice as large as the other bacterial enzymes and about the same size as the mouse enzyme, the only mammalian CMP-NeuAc synthetase is sequenced. The N-terminal half of the E. coli K1 sequence is highly homologous to other bacterial sequences, but the full-length sequence share a limited homology with the mouse sequence. We will crystallize the E. coli K1 CMP-NeuAc synthetase for structure determination. The structural differences of these functional homologues will be used to design drugs to target the bacterial enzymes specifically.
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