Structural Studies of <i>Escherichia coli</i> UDP-<i>N</i>-Acetylmuramate:<scp>l</scp>-Alanine Ligase

Hu, Jin; Emanuele, John J.; Fairman, Robert; Robertson, James G.; Hail, Mark E.; Ho, Hsu‐Tso; Falk, Paul; Villafranca, Joseph J.

doi:10.1021/bi952334k

Cited by 40 publications

(36 citation statements)

References 20 publications

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“…The UDP-MurNAc:L-Ala and UDP-MurNAc:Gly ligase activities were assayed as described by Liger et al (12). For this purpose, UDP-MurNAc was prepared by a two-step coupled enzymatic conversion of UDP-GlcNAc to UDP-MurNAc according to Jin et al (9) and identified through negative-ion fast atom bombardment-mass spectroscopy (FAB-MS) (the expected mass of 679 was observed) and nuclear magnetic resonance (NMR) (300 MHz). The following signals were clearly identified by 1 acid-1 M ammonium hydroxide (5:3) to separate the reaction product from unreacted amino acids.…”

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confidence: 99%

Comparison of the UDP- N -Acetylmuramate: l -Alanine Ligase Enzymes from Mycobacterium tuberculosis and Mycobacterium leprae

2000

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In the peptidoglycan of Mycobacterium leprae, L-alanine of the side chain is replaced by glycine. When expressed in Escherichia coli, MurC (UDP-N-acetyl-muramate:L-alanine ligase) of M. leprae showed K m and V max for L-alanine and glycine similar to those of Mycobacterium tuberculosis MurC, suggesting that another explanation should be sought for the presence of glycine.Some chemical differences exist in the peptidoglycan of Mycobacterium spp. compared to other bacteria (3). Mycobacterial muramic acid is thought to be glycolylated instead of acetylated (14). In the case of Mycobacterium leprae, the first amino acid in the tetrapeptide side chain of the peptidoglycan is Gly instead of L-Ala (5), as found in Mycobacterium tuberculosis and many other bacteria, implying that the M. leprae genome may encode a unique UDP-N-acetylmuramate:L-Ala/Gly (UDP-MurNAc:L-Ala/Gly) ligase (MurC) specific for the addition of Gly to UDP-MurNAc. These special structural features of mycobacterial peptidoglycan suggest the presence of unique enzymes that could be exploited as drug targets.The genes that encode MurC from several organisms have been sequenced (1,6,8,11,13), and the Escherichia coli MurC has been overexpressed and characterized (7, 11). However, the mycobacterial counterparts have not been studied. The availability of the genome sequences of M. tuberculosis (4) and M. leprae (ftp://ftp.sanger.ac.uk/pub/pathogens/leprae/) provides an opportunity to study the enzymes of these two pathogenic species, especially important in the case of M. leprae, which is not accessible to direct enzymatic study.murC genes of Mycobacterium. The complete sequence of the open reading frame of MLCB268.01c was revealed from the assembled genome sequence of M. leprae; it corresponds to bp 1084518 to 1086003. The resulting protein contains 595 amino acid residues with a theoretical molecular mass of 51 kDa, very similar to M. tuberculosis MurC (about 79% identity) but with only ϳ34% identity to E. coli MurC. Both Rv2152c (M. tuberculosis) and MLCB268.01 (M. leprae) are found within the mra cluster and contain eight of nine invariant amino acids (2) that align perfectly with known MurCs from other organisms (Fig. 1). However, the MurC homologs found outside the mra clusters (Rv3712 and MLCB2407.24c) have only ϳ22% identity with the putative MurCs found within the clusters, and four of the nine invariant amino acids either are absent or did not align.Cloning, expression, and purification of UDP-N-MurNAc: L-Ala ligase (MurC). Rv2152c and Rv3712 were amplified from M. tuberculosis H37Rv genomic DNA and cloned into the pET29aϩ vector (Novagen, Madison, Wis.) (16), yielding pSM201 and pSM203, respectively. The M. leprae MLCB268.01 and MLCB2407.24c genes were amplified from M. leprae genomic DNA and cloned into pET28aϩ and pET29aϩ, respectively, yielding pSM206 and pSM208, respectively (16,17). E. coli BL21(DE3) harboring plasmid pSM201, pSM203, pSM206, or pSM208 was grown in Luria-Bertani broth containing kanamycin, induced with isopropyl-␤-D-thiogal...

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confidence: 99%

Comparison of the UDP- N -Acetylmuramate: l -Alanine Ligase Enzymes from Mycobacterium tuberculosis and Mycobacterium leprae

2000

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“…Interestingly, Ec MurC (PDB 2f00) is also a dimer in the crystal structure but, in solution, displays a dynamic equilibrium between monomeric and dimeric forms. Both forms were shown to be active [16], [60]. The PFYG (residues 222–225) motif in Ec MurC (corresponding to PSTG, residues 232–235, in Pa Mpl), which is conserved in ∼50% of MurC proteins, is involved in dimerization.…”

Section: Resultsmentioning

confidence: 99%

Structure and Function of the First Full-Length Murein Peptide Ligase (Mpl) Cell Wall Recycling Protein

et al. 2011

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Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In Gram-negative bacteria, ∼30–60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships.

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“…The dimer interface is formed by the interaction of loops from the top of domain II of one molecule with loops from domain I of the second molecule and the most important interactions involve Phe223 and Tyr224 [83], however in M. tuberculosis the corresponding residues are not conserved (Pro218 and Gly219).…”

Section: Methodsmentioning

confidence: 99%

Structural and functional features of enzymes of Mycobacterium tuberculosis peptidoglycan biosynthesis as targets for drug development

et al. 2015

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SUMMARY Tuberculosis (TB) is the second leading cause of human mortality from infectious diseases worldwide. The WHO reported 1.3 million deaths and 8.6 million new cases of TB in 2012. Mycobacterium tuberculosis (M. tuberculosis), the infectious bacteria that causes TB, is encapsulated by a thick and robust cell wall. The innermost segment of the cell wall is comprised of peptidoglycan, a layer that is required for survival and growth of the pathogen. Enzymes that catalyse biosynthesis of the peptidoglycan are essential and are therefore attractive targets for discovery of novel antibiotics as humans lack similar enzymes making it possible to selectively target bacteria only. In this paper, we have reviewed the structures and functions of enzymes GlmS, GlmM, GlmU, MurA, MurB, MurC, MurD, MurE and MurF from M. tuberculosis that are involved in peptidoglycan biosynthesis. In addition, we report homology modelled 3D structures of those key enzymes from M. tuberculosis of which the structures are still unknown. We demonstrated that natural substrates can be successfully docked into the active sites of the GlmS and GlmU respectively. It is therefore expected that the models and the data provided herein will facilitate translational research to develop new drugs to treat TB.

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Structural Studies of Escherichia coli UDP-N-Acetylmuramate:l-Alanine Ligase

Cited by 40 publications

References 20 publications

Comparison of the UDP- N -Acetylmuramate: l -Alanine Ligase Enzymes from Mycobacterium tuberculosis and Mycobacterium leprae

Comparison of the UDP- N -Acetylmuramate: l -Alanine Ligase Enzymes from Mycobacterium tuberculosis and Mycobacterium leprae

Structure and Function of the First Full-Length Murein Peptide Ligase (Mpl) Cell Wall Recycling Protein

Structural and functional features of enzymes of Mycobacterium tuberculosis peptidoglycan biosynthesis as targets for drug development

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