Exploitation of structural and regulatory diversity in glutamate racemases

Abstract

Glutamate racemase is an enzyme essential to the bacterial cell wall biosynthesis pathway, and has therefore been considered as a target for antibacterial drug discovery. We characterized the glutamate racemases of several pathogenic bacteria using structural and biochemical approaches. Here we describe three distinct mechanisms of regulation for the family of glutamate racemases: allosteric activation by metabolic precursors, kinetic regulation through substrate inhibition, and D-glutamate recycling using a d-amino acid transaminase. In a search for selective inhibitors, we identified a series of uncompetitive inhibitors specifically targeting Helicobacter pylori glutamate racemase that bind to a cryptic allosteric site, and used these inhibitors to probe the mechanistic and dynamic features of the enzyme. These structural, kinetic and mutational studies provide insight into the physiological regulation of these essential enzymes and provide a basis for designing narrow-spectrum antimicrobial agents.

Evaluation of new antimicrobial agents on Bacillus spp. strains: docking affinity and in vitro inhibition of glutamate-racemase

“…Likewise, the natural substrate was close to the following residues of RacE2: Ser12, Tyr43, Asp11, Thr186, Thr76, Asn75, Gly44, Pro42 and Val149. These results are in agreement with reports in the literature 3,10,21 . Regarding l-GM, in the interaction with RacE1 it was close to Arg43, Thr124 and Thr153.…”

“…Various studies have reported that some glutamic acid derivatives are very selective for RacE and have potent antimicrobial activity 21,22 . In the current contribution, three glutamic acid derivatives, two analogues of l-glutamic acid and one of d-glutamic acid, were synthesized and tested as RacE inhibitors.…”

Moonlighting function of glutamate racemase from Mycobacterium tuberculosis: racemization and DNA gyrase inhibition are two independent activities of the enzyme

“…MurI is an attractive target for designing new antibacterial drugs as it provides D-glutamate, an important component for peptidoglycan biosynthesis (Lundqvist et al, 2007). Overexpression of M. tuberculosis MurI in soluble form and also in large quantities described here Cultures of M. smegmatis harbouring either the vector pJAM2 or pJAM2-mtmurI constructs (wild-type or double mutant) were grown in Middlebrook 7H9 broth to mid-exponential phase at 37 u C and then poured onto soft agar plates to form a lawn.…”

Determination of Antibiotic Hypersensitivity among 4,000 Single-Gene-Knockout Mutants of Escherichia coli

“…While these lines of investigation show great promise, additional approaches are constantly being sought to yield a new generation of useful antimicrobial compounds. For instance, focusing on species-specific antibiotics rather than broad-spectrum antibiotics can result in important new agents (38), as could targeting bacterial transcription factors (5) or different processes, such as Holliday junction processing (21,31) and quorum sensing (24), or even targeting host factors that support pathogen growth (33). Another approach, examined here, involves potentiating existing antibiotics by identifying targets for increasing susceptibility to specific antimicrobials.…”