Structural genomics of highly conserved microbial genes of unknown function in search of new antibacterial targets

J Struct Funct Genomics. 2003;4(2-3):141-57. doi: 10.1023/a:1026177202925.

Abstract

With more than 100 antibacterial drugs at our disposal in the 1980's, the problem of bacterial infection was considered solved. Today, however, most hospital infections are insensitive to several classes of antibacterial drugs, and deadly strains of Staphylococcus aureus resistant to vancomycin--the last resort antibiotic--have recently begin to appear. Other life-threatening microbes, such as Enterococcus faecalis and Mycobacterium tuberculosis are already able to resist every available antibiotic. There is thus an urgent, and continuous need for new, preferably large-spectrum, antibacterial molecules, ideally targeting new biochemical pathways. Here we report on the progress of our structural genomics program aiming at the discovery of new antibacterial gene targets among evolutionary conserved genes of uncharacterized function. A series of bioinformatic and comparative genomics analyses were used to identify a set of 221 candidate genes common to Gram-positive and Gram-negative bacteria. These genes were split between two laboratories. They are now submitted to a systematic 3-D structure determination protocol including cloning, protein expression and purification, crystallization, X-ray diffraction, structure interpretation, and function prediction. We describe here our strategies for the 111 genes processed in our laboratory. Bioinformatics is used at most stages of the production process and out of 111 genes processed--and 17 months into the project--108 have been successfully cloned, 103 have exhibited detectable expression, 84 have led to the production of soluble protein, 46 have been purified, 12 have led to usable crystals, and 7 structures have been determined.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acid Anhydride Hydrolases / chemistry
  • Alcohol Oxidoreductases / chemistry
  • Amino Acid Sequence
  • Anti-Bacterial Agents / pharmacology*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Binding Sites
  • Carrier Proteins / chemistry
  • Conserved Sequence
  • Crystallography, X-Ray
  • Drug Design*
  • Endopeptidases / chemistry
  • Escherichia coli / genetics
  • Gene Expression
  • Genes, Bacterial*
  • Genomics / methods*
  • Models, Molecular
  • Molecular Sequence Data
  • Oxidoreductases / chemistry
  • Phylogeny
  • Polymerase Chain Reaction / methods
  • Protein Conformation
  • Sequence Alignment

Substances

  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Carrier Proteins
  • Oxidoreductases
  • Alcohol Oxidoreductases
  • Shikimate dehydrogenase
  • Endopeptidases
  • Acid Anhydride Hydrolases