Glutamate racemase from Mycobacterium tuberculosis inhibits DNA gyrase by affecting its DNA-binding

Abstract

Glutamate racemase (MurI) catalyses the conversion of l-glutamate to d-glutamate, an important component of the bacterial cell wall. MurI from Escherichia coli inhibits DNA gyrase in presence of the peptidoglycan precursor. Amongst the two-glutamate racemases found in Bacillus subtilis, only one inhibits gyrase, in absence of the precursor. Mycobacterium tuberculosis has a single gene encoding glutamate racemase. Action of M.tuberculosis MurI on DNA gyrase activity has been examined and its mode of action elucidated. We demonstrate that mycobacterial MurI inhibits DNA gyrase activity, in addition to its precursor independent racemization function. The inhibition is not species-specific as E.coli gyrase is also inhibited but is enzyme-specific as topoisomerase I activity remains unaltered. The mechanism of inhibition is different from other well-known gyrase inhibitors. MurI binds to GyrA subunit of the enzyme leading to a decrease in DNA-binding of the holoenzyme. The sequestration of the gyrase by MurI results in inhibition of all reactions catalysed by DNA gyrase. MurI is thus not a typical potent inhibitor of DNA gyrase and instead its role could be in modulation of the gyrase activity.

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

“…Escherichia coli strains DH5a and BL26(DE3) were used for cloning and overexpression, respectively, of Mycobacterium tuberculosis MurI mutants. The murI gene of M. tuberculosis was mobilized in the pJAM2 vector (Triccas et al, 1998) from the pET11d-murI construct (Sengupta et al, 2006) to generate pJAM2-mtmurI. Mycobacterium smegmatis mc 2 155 was used to express MurI in a mycobacterial system.…”

“…The MurI mutants were overexpressed from pET20b constructs in E. coli BL26(DE3). The proteins were purified as described previously (Sengupta et al, 2006).…”

“…The racemization activity of MurI was assessed as described previously . MurI samples were incubated in the presence of D-glutamate (Sigma) and then rapidly heated to inactivate the enzyme and assayed for Lglutamate (Sigma) using NAD + /L-glutamate dehydrogenase (Sigma) (Sengupta et al, 2006). In another independent assay, varying concentrations of MurI (wild-type, C75S, C185S and C75SC185S double mutant) were incubated in the presence of either D-or Lglutamate substrate (10 mM) in a buffer containing 10 mM Tris/HCl, pH 8.0, and 4 mM b-mercaptoethanol at 30 uC for 30 min.…”

“…In our previous studies we established the mechanism of inhibition of DNA gyrase by MurI (Sengupta et al, 2006). MurI from M. tuberculosis interacts with the GyrA subunit of DNA gyrase, which is the primary DNA-binding subunit of the holoenzyme, also responsible for DNA cleavage and religation (Sengupta et al, 2006).…”

“…Subsequently, YrpC, one of the two glutamate racemases from Bacillus subtilis, was shown to influence the activity of DNA gyrase in precursorindependent fashion (Ashiuchi et al, 2003). MurI from Mycobacterium tuberculosis was shown to inhibit DNA gyrase by preventing DNA-binding activity of the enzyme (Sengupta et al, 2006). Gyrase inhibition thus seems to be an additional property of these enzymes.…”