Glutamate Racemase From Mycobacterium tuberculosis: New Genetic and Structural Insights Into a Target for Antituberculosis Drug Design

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Background. Glutamate racemase, the enzyme that provides d-glutamate for bacterial cell well biosynthesis, has been the target of several anti-microbial drug development programs. Recent genetic work suggests it could be a promising target as well for anti-tuberculosis drug design. As Mycobacterium tuberculosis remains a deadly human pathogen with emerging drug-resistance, there is a pressing need for new anti-tuberculosis therapies. Methods. Deletion mutagenesis work on murI has been previously reported by us in M. smegmatis. This work has been extended by determining the structure and basic regulation of the murI operon by analyzing cDNA libraries for intragenic transcripts and by studying expression under conditions of high and low osmolality and in the presence of supplemental amino acids. Structural biology studies of glutamate racemase, carried out by cloning, expression and crystallization of purified enzyme, have been extended through kinetic and biophysical analysis of wild-type and mutant enzymes. Results. Previous work carried out in our laboratory and others have shown the murI gene to be essential in mycobacteria. Our operon work has established that murI is part of a multi-gene operator in M. smegmatis and has been found to be constitutively expressed. Crystal structures of glutamate racemase from M. tuberculosis and M. smegmatis have been analyzed at 2.3Å and 1.8Å, respectively, and the interactions of the active site residues with the substrate, d-glutamate, have been confirmed. Both mycobacterial enzymes have been found to exist as a tight dimer with a unique dimer interface unreported in other glutamate racemase structures. This dimer interface has been mutated by us to allow for kinetic characterization. The enzyme possesses binding pockets at the active site as well as adjacent to the active that that could provide a promising target for future structure-based anti-tuberculosis drug design efforts. Conclusion. Glutamate racemase has unique genetic and structural features and holds promise as a template for future structure-based anti-tuberculosis drug design efforts. Disclosures. All authors: No reported disclosures.