UDP-N-ACETYLMURAMYL-L-ALANINE FUNCTIONS AS AN ACTIVATOR IN THE REGULATION OF THE ESCHERICHIA-COLI GLUTAMATE RACEMASE ACTIVITY

D-Glutamate is an essential component of the bacterial peptidoglycan. In Escherichia coli, the biosynthesis of D-glutamate is catalyzed by a glutamate racemase (encoded by the dga gene) and is regulated by UDP-N-acetylmuramyl-L-alanine [Doublet et al. (1994) Biochemistry 33, 5285], a bacterial peptidoglycan subunit precursor. Investigation was conducted to elucidate the interaction between the enzyme and its regulator. Whole and N-terminal truncated enzymes, encoded by individual constructs containing either a full-length or an N-terminal truncated dga gene, were evaluated. In the absence of the regulator, the purified whole enzyme showed a low-level basal racemase activity for which a K-m value of 18.9 mM and a V-max of 0.4 mu mol/(min . mg) were determined, using D-glutamate as the substrate. Using the same substrate, in the presence of 6.5 mu M UDP-N-acetylmuramyl-L-alanine, a K-m value of 4.2 mM and a V-max of 34 mu mol/(min . mg) were measured. Similar kinetic parameters for the activated enzyme were obtained using L-glutamate as the substrate. The N-terminal truncated E. coli enzyme, with a 21 amino acid region removed, is similar in size to the Pediococcus pentosaceus glutamate racemase. Effects of the regulator on the full-length and the N-terminal truncated enzyme in the dialyzed cell lysate were compared. A host cell line, E. coli WM335 Delta rec Delta, containing a nonfunctional chromosomal dga gene was used to minimize the background interference. With 6.5 mu M regulator added, the N-terminal truncated enzyme displayed a loss of more than 80% of the activity compared to the full-length enzyme. This loss of activation effect was repeated in the presence of 0.65 and 65 mu M regulator. Evaluation of the Pediococcus enzyme revealed no detectable effect exerted by UDP-N-acetylmuramyl-L-alanine. The activated E. coli racemase is at least as efficient as the Pediococcus racemase. These results indicate that the regulator is unique to the E. coli enzyme and the longer N-terminal region of the enzyme is necessary for its optimal activation. This regulation suggests that a more stringent control of the cytoplasmic level of D-glutamate may be required in Gram-negative bacteria. H-1 NMR studies showed that the UDP-N-acetylmuramyl-L-alanine-activated E. coli racemase catalyzed the exchange of the solvent H-2 With the C-2 H-1 of the substrate. Site-directed mutagenesis carried out at either of the two conserved cysteine (C96T and C208T) residues resulted in inactivation of the enzyme. These two cysteine residues may be involved in the extraction and readdition of the C-2 proton in the racemization process. The UDP-Nacetylmuramyl-L-alanine-activated E. coli glutamate racemase appears to follow a deprotonation/reprotonation mechanism as reported for Lactobacilli glutamate racemase. However, the latter was reported not to be regulated by this regulator and to have significantly higher efficiency.