Isolation and characterization of uridine diphosphate-N-glycolylmuramyl-L-alanyl-γ-D-glutamyl-meso-α,α′-diaminopimelic acid from Mycobacterium tuberculosis

Takayama, Kuni; David, Hugo L.; Wang, Lynn; Goldman, Dexter S.

doi:10.1016/0006-291x(70)90749-7

Cited by 32 publications

(16 citation statements)

References 16 publications

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“…Previously, this problem had been addressed by treating bacterial cells with peptidoglycan biosynthesis inhibitors to allow accumulation of the precursors, thus enabling isolation of tangible quantities for analysis (3,17). Studies of the biosynthetic precursor UDP-Nacylmuramyl-tripeptide from M. tuberculosis and M. phlei (28,33) determined that all the muramic acid residues were, apparently, completely NGlyc substituted. Since UDP-MurNGlyctripeptide is the immediate precursor of the UDP-MurNGlycpentapeptide, all of the UDP-N-acylmuramyl-pentapeptides should be NGlyc substituted, as has been the supposition (5,7,18).…”

Section: Discussionmentioning

confidence: 99%

“…However, the peptidoglycan of mycobacteria contains a variety of reported modifications including, invariably, an N-glycolyl (NGlyc) instead of an N-acetyl (NAc) function on the muramic acid (Mur), amidation of the carboxylic acids, and additional glycine or serine residues (18,20). The peptidoglycan biosynthetic pathway of E. coli has been well studied (34,35), and the mycobacterial pathway is assumed to be the same, based on limited biochemical analysis of some years ago (25,28,33) and more recent comparative genomics (2,5,6,21). Analysis of the various precursors of peptidoglycan biosynthesis in Mycobacterium spp.…”

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confidence: 99%

“…Because of the relatively low abundance of these precursors in untreated cells, drug treatment was used in order to obtain sufficient amounts for chemical characterization in mycobacterial studies. An incomplete precursor, UDP-Nglycolylmuramyl-L-alanyl-D-glutamyl-diaminopimelic acid (UDP-MurNGlyc-tripeptide) was isolated and analyzed from M. tuberculosis and Mycobacterium phlei following accumulation after treatment with D-cycloserine (28,33). During a comparative study, we observed that the muramic acid residues isolated from mature peptidoglycan of M. tuberculosis and Mycobacterium smegmatis were a mixture of the N-acetyl and N-glycolyl derivatives, not solely the N-glycolylated product as generally reported (4,19,20).…”

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N Glycolylation of the Nucleotide Precursors of Peptidoglycan Biosynthesis of Mycobacterium spp. Is Altered by Drug Treatment

et al. 2005

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The peptidoglycan of Mycobacterium spp. reportedly has some unique features, including the occurrence of N-glycolylmuramic rather than N-acetylmuramic acid. However, very little is known of the actual biosynthesis of mycobacterial peptidoglycan, including the extent and origin of N glycolylation. In the present work, we have isolated and analyzed muramic acid residues located in peptidoglycan and UDP-linked precursors of peptidoglycan from Mycobacterium tuberculosis and Mycobacterium smegmatis. The muramic acid residues isolated from the mature peptidoglycan of both species were shown to be a mixture of the N-acetyl and N-glycolyl derivatives, not solely the N-glycolylated product as generally reported. The isolated UDP-linked N-acylmuramyl-pentapeptide precursor molecules also contain a mixture of N-acetyl and N-glycolyl muramyl residues in apparent contrast to previous observations in which the precursors isolated after treatment with D The peptidoglycan of Mycobacterium spp. is classified as A1␥, unmodified peptidoglycan, as is that of Escherichia coli and many other bacterial species (32). However, the peptidoglycan of mycobacteria contains a variety of reported modifications including, invariably, an N-glycolyl (NGlyc) instead of an N-acetyl (NAc) function on the muramic acid (Mur), amidation of the carboxylic acids, and additional glycine or serine residues (18,20). The peptidoglycan biosynthetic pathway of E. coli has been well studied (34,35), and the mycobacterial pathway is assumed to be the same, based on limited biochemical analysis of some years ago (25, 28, 33) and more recent comparative genomics (2, 5, 6, 21). Analysis of the various precursors of peptidoglycan biosynthesis in Mycobacterium spp. could provide important information on the biosynthetic origin of these modifications and the notable refractoriness of Mycobacterium tuberculosis to ␤-lactam antibiotics and help in the search for alternative drug regimens for the treatment of multiple-drug-resistant forms of tuberculosis (10, 16).UDP-N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine (UDP-MurNAc-pentapeptide) is the final cytosolic precursor of peptidoglycan biosynthesis in E. coli. Because of the relatively low abundance of these precursors in untreated cells, drug treatment was used in order to obtain sufficient amounts for chemical characterization in mycobacterial studies. An incomplete precursor, UDP-Nglycolylmuramyl-L-alanyl-D-glutamyl-diaminopimelic acid (UDP-MurNGlyc-tripeptide) was isolated and analyzed from M. tuberculosis and Mycobacterium phlei following accumulation after treatment with D-cycloserine (28, 33). During a comparative study, we observed that the muramic acid residues isolated from mature peptidoglycan of M. tuberculosis and Mycobacterium smegmatis were a mixture of the N-acetyl and N-glycolyl derivatives, not solely the N-glycolylated product as generally reported (4,19,20). Since the nucleotide-linked precursors should reflect the nature of mature peptidoglycan, we hypothesized that the...

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Section: Discussionmentioning

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N Glycolylation of the Nucleotide Precursors of Peptidoglycan Biosynthesis of Mycobacterium spp. Is Altered by Drug Treatment

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“…Previous studies used the drug D-cycloserine, which inhibits D-Ala-D-Ala ligase, to block incorporation of PG precursors into the PG in order to generate sufficient precursor material for analysis. They found that all muramic acids were N glycolylated (11,233,315,399). Recently, using a technique that does not require blocking PG synthesis, researchers found that mycobacterial PG contains both N-glycolyl and N-acetyl modifications (254).…”

Section: Cell Wall Components What Is the Structure Of The Mycobactermentioning

confidence: 99%

Bacterial Growth and Cell Division: a Mycobacterial Perspective

Hett

Rubín

2008

Microbiol Mol Biol Rev

342

293

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SUMMARY The genus Mycobacterium is best known for its two major pathogenic species, M. tuberculosis and M. leprae, the causative agents of two of the world's oldest diseases, tuberculosis and leprosy, respectively. M. tuberculosis kills approximately two million people each year and is thought to latently infect one-third of the world's population. One of the most remarkable features of the nonsporulating M. tuberculosis is its ability to remain dormant within an individual for decades before reactivating into active tuberculosis. Thus, control of cell division is a critical part of the disease. The mycobacterial cell wall has unique characteristics and is impermeable to a number of compounds, a feature in part responsible for inherent resistance to numerous drugs. The complexity of the cell wall represents a challenge to the organism, requiring specialized mechanisms to allow cell division to occur. Besides these mycobacterial specializations, all bacteria face some common challenges when they divide. First, they must maintain their normal architecture during and after cell division. In the case of mycobacteria, that means synthesizing the many layers of complex cell wall and maintaining their rod shape. Second, they need to coordinate synthesis and breakdown of cell wall components to maintain integrity throughout division. Finally, they need to regulate cell division in response to environmental stimuli. Here we discuss these challenges and the mechanisms that mycobacteria employ to meet them. Because these organisms are difficult to study, in many cases we extrapolate from information known for gram-negative bacteria or more closely related GC-rich gram-positive organisms.

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“…In addition, the modifications found in the mature peptidoglycans of some bacteria occur at the lipid-linked intermediate level (6,15,18,19,23,31,34), but it has been reported that the oxidation of the N-acetyl function of the N-acetylmuramic acid in mycobacteria occurs at the nucleotide level (32). Therefore, the objectives of the present study were to identify which of the three candidate lipid carrier molecules are utilized by M. smegmatis in the formation of lipid I and II and to determine if the modifications in the mature mycobacterial peptidoglycan occur on lipid-linked intermediates.…”

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Mycobacterial Lipid II Is Composed of a Complex Mixture of Modified Muramyl and Peptide Moieties Linked to Decaprenyl Phosphate

et al. 2005

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Structural analysis of compounds identified as lipid I and II fromMycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N-acetylmuramyl-L-alanine-D-glutamate-meso-diaminopimelic acid-D-alanyl-D-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N-acetylmuramic acid, N-glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal D-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.Peptidoglycans are essential components of bacterial cell walls, providing both mechanical strength and shape. The basic structure of peptidoglycan is common among most of the eubacteria, consisting of glycan chains composed of alternating units of ␤134-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). The lactyl group of the MurNAc residue usually carries a short peptide (L-Ala-DGlu-meso-diaminopimelic acid [DAP]-D-Ala-D-Ala) that forms bonds with the peptide side chains of neighboring glycan strands, thus providing mechanical strength (35). Although the basic structure of peptidoglycan remains the same among eubacterial species, there are significant variations (16, 29). The structure of the mycobacterial peptidoglycan is an example of such divergence (16,27,40). The glycan chains of mycobacterial peptidoglycan are composed of alternating units of ␤134-linked GlcNAc and N-glycolylmuramic acid (MurNGlyc) in which the N-acetyl function has been oxidized to an N-glycolyl function. The carbon-6 position of some of the muramic acid residues forms a phosphodiester bond with the ␣-L-rhamnopyranose-(133)-␣-D-GlcNAc(1-P) linker region of the galactan chain of the arabinogalactan (24), and about a third of the peptide cross bridges occur between the carboxyl group of the L-center of one DAP residue and the amino group of the D-center of another DAP residue, forming a L,D-cross-link (40); the rest of the cross-links are between the carbo...

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Isolation and characterization of uridine diphosphate-N-glycolylmuramyl-L-alanyl-γ-D-glutamyl-meso-α,α′-diaminopimelic acid from Mycobacterium tuberculosis

Cited by 32 publications

References 16 publications

N Glycolylation of the Nucleotide Precursors of Peptidoglycan Biosynthesis of Mycobacterium spp. Is Altered by Drug Treatment

N Glycolylation of the Nucleotide Precursors of Peptidoglycan Biosynthesis of Mycobacterium spp. Is Altered by Drug Treatment

Bacterial Growth and Cell Division: a Mycobacterial Perspective

Mycobacterial Lipid II Is Composed of a Complex Mixture of Modified Muramyl and Peptide Moieties Linked to Decaprenyl Phosphate

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