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

Abstract

UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase is a cytoplasmic enzyme that catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor UDP-N-acetylmuramoyl-L-alanyl-D-glutamate in the biosynthesis of bacterial cell-wall peptidoglycan. The crystal structure of the Escherichia coli enzyme in the presence of the final product of the enzymatic reaction, UDP-MurNAc-L-Ala-␥-D-Glu-meso-A 2 pm, has been solved to 2.0 Å resolution. Phase information was obtained by multiwavelength anomalous dispersion using the K shell edge of selenium. The protein consists of three domains, two of which have a topology reminiscent of the equivalent domain found in the already established three-dimensional structure of the UDP-N-acetylmuramoyl-L-alanine: D-glutamateligase (MurD) ligase, which catalyzes the immediate previous step of incorporation of D-glutamic acid in the biosynthesis of the peptidoglycan precursor. The refined model reveals the binding site for UDP-MurNAc-LAla-␥-D-Glu-meso-A 2 pm, and comparison with the six known MurD structures allowed the identification of residues involved in the enzymatic mechanism. Interestingly, during refinement, an excess of electron density was observed, leading to the conclusion that, as in MurD, a carbamylated lysine residue is present in the active site. In addition, the structural determinant responsible for the selection of the amino acid to be added to the nucleotide precursor was identified.Peptidoglycan, the polymeric mesh of the bacterial cell wall, plays a critical role in protecting bacteria against osmotic lysis. It consists of linear repeating disaccharide chains cross-linked by short peptide bridges. During the cytoplasmic steps involved in the biosynthesis of the peptidoglycan precursor, four ADPforming ligases (namely the Mur ligases) catalyze the assembly of the peptide moiety by the successive addition of L-alanine, D-glutamate, diaminopimelic acid, or L-lysine, and, finally, dipeptide D-alanyl-D-alanine to UDP-N-acetylmuramic acid (1, 2). Because all these enzymes are essential for cell viability, they are attractive targets for antibacterial chemotherapy. In Escherichia coli, these ligases are the products of the murC, murD, murE, and murF genes, located in the mra region (3). Sequence comparison of the four E. coli Mur ligases shows several homologous regions, suggesting that these enzymes may be evolutionarily related and may use similar enzymatic mechanisms (4 -6). In earlier publications, we reported the structure of UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD), 1 both in the native form and complexed with substrates (7-9). MurD consists of three domains with topologies reminiscent of a nucleotide-binding fold; the N-and Cterminal domains have a dinucleotide-binding fold (the Rossmann fold), and the central domain displays a mononucleotidebinding fold, also seen in ATP-binding proteins. A comparison of six MurD structures reveals that large C-terminal rotation, loop rearrangement, and subdomain movements occur upon subs...

The MurE Synthetase from Thermotoga maritima Is Endowed with an Unusual d-Lysine Adding Activity

“…The T. maritima murE gene was amplified by PCR from the chromosome of strain MSB8; for this purpose, two primers containing a BspHI site (shown in bold type) 5Ј to the initiation codon (underlined), 5Ј-TCTA-TCATGAATATATCAACTATCGTGTCG-3Ј, and a BamHI site (in bold type) 3Ј to the gene without its stop codon, 5Ј-AGATGGATCC-TTGGGCGTATTTCCTCCCCTTCAGC-3Ј, respectively, were employed. The amplified material was digested by BspHI and BamHI and inserted between the compatible NcoI and BglII sites of vectors pET2160 (T7 promoter) (8) and pTrcHis60 (trc promoter) (21), generating plasmids pABO6 and pABO7, respectively, that code for T. maritima MurE with an Arg-Ser-(His) 6 C-terminal extension. The constructions were verified by DNA sequencing (MWG-Biotech).…”

“…The D-lysine-containing UDP-MurNAc-tripeptide was also produced by MurE from E. coli (6). In this case, the buffer was 0.1 M TrisHCl, pH 8.6, and the MgCl 2 concentration was 100 mM.…”

“…Therefore, MurE must be endowed with a high specificity to select the "right" amino acid among closely related amino acids that coexist within the cell. Recently, the crystallization of MurE from Escherichia coli allowed Gordon et al (6) to decipher the structural bases for this high specificity.…”

Complete genome sequence of the rifamycin SV-producing Amycolatopsis mediterranei U32 revealed its genetic characteristics in phylogeny and metabolism

“…Furthermore, genes involved in incorporating arabinose into the cell wall, characterized in Mycobacterium [21], can be found in the genomes of A. mediterranei, N. farcinica, and S. erythraea, but not in most of the Streptomyces species (Supplementary information, Table S5). In addition, the MurE ligase, which ligates specific amino acids [22] to the lateral chain of peptidoglycan, was analyzed phylogenetically within actinomycetes (Supplementary information, Figure S2). The MurE of A. mediterranei, and of the closely related S. erythraea, was clustered within a big clade composed of the meso- npg DAP-containing actinomycetes, strongly supporting the taxonomic character of A. mediterranei, that is, that meso-DAP rather than LL-DAP is used as the substrate to synthesize its cell wall.…”

An Integration of Interdisciplinary Translational Research in Anti-TB Drug Discovery: Out of the University Research Laboratories to Combat Mycobacterium tuberculosis

“…These enzymes contribute to the synthesis of UDP N-acetyl muramoyl pentapeptide through a sequential peptide-ligase reaction by using UDP-MurNAc, ATP and different amino acid substrates. Crystal structures of the recombinant Mur ligases in E. coli have been determined [19][20][21][22]. All these Mur ligases have been found to be essential in a number of pathogenic bacteria.…”