Specificity of the uridine‐diphosphate‐N‐acetylmuramyl‐l‐alanyl‐d‐glutamate: meso‐2,6‐diaminopimelate synthetase from Escherichia coli

Abstract

To investigate the specificity of the urid~ne-d~phosphate-~-acetylmuramyl-~-alanyl-~-g~utamate: meso-2,6-diaminopimelate synthetase, various compounds mimicking more or less different parts of the UDP-MurNAc-1.-Ala-D-Glu substrate were prepared. Their size ranged from that of uridine or L-Ala-D-Glu to that of the whole nucleotide substrate. Chemical synthesis led to Nu-acyl-dipeptides, in which the acyl group mimicked the MurNAc moiety, and to glycopeptides MurNAc(a or P-Me)-L-Ala-u-Glu, in which the anomeric function is blocked. Partial degradation or chemical modification of the substrate UDP-MurNAc-L-Ala-D-Glu afforded : MurOHNAc-L-Alau-Glu, P'-MurNAc-L-Ala-Ii-Glu, and DDP-MurNAc-L-Ala-u-Glu (DDP = dihydrouridine-diphosphate). All these compounds were tested as substrates or (and) inhibitors of the reaction catalyzed by the A2pm-adding enzyme, which, after partial purification, was obtained in two active forms. Among the compounds tested as substrates, only DDP-MurNAc-L-Ala-D-Glu was a good one. The K , for this compound was 97 pM versus 55 pM for the natural substrate. Among the various compounds tested as inhibitors, only P'-MurNAc-L-Ala-D-Clu and MurNAc(a or P-Me)-L-Ala-u-Gh had a significant inhibitory effect at 1 mM. Apparently, no particular portion of the molecule is predominantly responsible for its recognition by the enzyme. In other words, multiple sites located over the whole molecule are required for a proper recognition and determine the high specificity of this activity. Therefore, to obtain efficient competitive inhibitors it is necessary to synthesize molecules very similar in size and structure to the natural substrate.The cytoplasmic steps of the biosynthesis of bacterial cell wall peptidoglycan involve a series of uridine nucleotide precursors including, in particular, UDP-N-acetylmuramic acid and UDP-MurNAc-peptides [ 11. These latter compounds are intermediates in the reaction sequence leading to the formation of UDP-MurNAc-pentapeptide from UDP-MurNAc by the stepwise addition of L-alanine, D-glUtamiC acid, generally diaminopimelic acid or L-lysine, and D-alanyl-D-alanine. Each step is catalysed by a particular synthetase, the specificity of which has been examined in a few instances [l]. One approach to the systematic study of the specificity of such enzymes is to prepare substrate analogues and to test them as substrates or (and) inhibitors. A better understanding of the specificity of these synthetases would in turn be useful for the design and synthesis of more specific inhibitors. Owing to the fact that the reactions these enzymes catalyse are only encountered Abbreviations. Aad, 2-aminoadipic acid; A2pm, 2,6-diaminopimelic acid; Bzi, benzylidene; Bzl, benzyl; H4furan, tetrahydrofuran; HPLC, high-performance liquid chromatography; DDP, dihydrouridine-.5'-diphosphate; Lac, lactyl; Me, methyl; Meb, methylbuturyl; MeOH, methanol; Mur, muramic acid; MurOH, muramicitol [2-amino-3-0-(~-1 -carboxyethyl)-2-deoxy-~-glucito1]; NAc, Nacetyl; Nps, 2-nitrophenylsulfenyl; Pr, propionyl; Su, 1 -suc...

Crystal Structure of UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-Diaminopimelate Ligase from Escherichia Coli

“…In addition, it is inserted between a salt bridge (between Asp 27 and Arg 29 ) and Tyr 50 . Interestingly, C 5 and C 6 of the pyrimidine ring are exposed to the solvent, explaining how dihydrouridine can replace uridine in the nucleotide substrate with little effect on the K m (97 M versus 55 M for the natural substrate) (43).…”

“…The protein-phosphate interactions are made mainly through hydrogen bonds with main chain nitrogens, the ␣-phosphate oxygens forming hydrogen bonds with Gln 44 and Ala 45 , and the ␤-phosphate oxygens with His 43 . One of the ␤-phosphate oxygens forms hydrogen bonds with ND1 of His 43 ; the latter is the only residue to balance the negative charges of the pyrophosphate.…”

Genome Sequence of Kitasatospora setae NBRC 14216T: An Evolutionary Snapshot of the Family Streptomycetaceae

“…50 (ii) Incorporation of DAP into peptidoglycan: the addition of DAP to UDP- N -acetylmuramyl- l -alanyl- d -glutamate precursor is catalyzed by an enzyme encoded by murE . 51 …”

Exploiting current understanding of antibiotic action for discovery of new drugs

“…These have no eukaryotic counterpart and thereby provide opportunities for discovering selective bacterial inhibitors (van Heijenoort 1996). Initially, attempts to discover specific inhibitors of these enzymes focused upon the preparation of analogues similar in size and structure to the natural substrates (Aboghalia et al . 1985).…”

“…1985). Such substrate analogues, however, are not readily transported into bacteria and therefore exhibit poor antibacterial activity (Aboghalia et al . 1985).…”