Amino Acid Residues Recognizing Isomeric Glutamate Substrates in UDP‑N‑acetylmuramic acid‑l‑alanine-glutamate Synthetases

We recently revealed that a previously unknown pathway for peptidoglycan biosynthesis operates in some microorganisms, including Xanthomonas oryzae. It involves two enzymes, MurD2 and MurL, which catalyze the ligation of l-glutamate (l-Glu) to UDP-N-acetylmuramic acid-l-alanine and the epimerization of the terminal l-Glu of the product, respectively. MurD2 of X. oryzae possesses a 26% identity with MurD of Escherichia coli (MurDec), which ligates d-Glu to UDP-N-acetylmuramic acid-l-alanine. To understand how X. oryzae MurD2 recognizes the isomer substrate, we estimated its structure based on that of MurDec during docking simulations. Several amino acid residues, which may be responsible for l-Glu recognition, were replaced with their corresponding amino acid residues in MurDec. Consequently, we obtained a mutated MurD2 enzyme that contained two amino acid substitutions and accepted only d-Glu as the substrate. We next tried to convert the substrate specificity of MurDec using the same strategy, but the mutant enzyme still accepted only d-Glu. Then, MurD of Streptococcus mutans (MurDsm), which possesses the key amino acid residue for l-Glu recognition identified in MurD2, was used for random screenings of mutant enzymes accepting l-Glu. We obtained a mutated MurDsm that had one amino acid substitution and slightly accepted l-Glu. A mutated MurDec possessing the corresponding one amino acid substitution also accepted l-Glu. Thus, we revealed that a few amino acid residues in MurD/MurD2 might control the acceptability of substrates with different stereochemistries.