Intrinsic Lipid Preferences and Kinetic Mechanism of <i>Escherichia coli</i> MurG

Chen, Lan; Men, Hongbin; Ha, Sha; Ye, Xiang‐Yang; Brunner, Livia; Hu, Yanan; Walker, Suzanne

doi:10.1021/bi0256678

Cited by 79 publications

(89 citation statements)

References 52 publications

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“…Altogether, our experimental data support a model wherein flexibility and conformational transitions confer upon PimA the adaptability to the donor and acceptor substrates required for catalysis (28). In this regard, PimA thus seems to follow an ordered mechanism similar that reported for other GT-B enzymes (29,30).…”

Section: Pim 2 Biosynthesissupporting

confidence: 83%

Molecular Basis of Phosphatidyl-myo-inositol Mannoside Biosynthesis and Regulation in Mycobacteria

Guerin

Korduláková

Alzari

et al. 2010

Journal of Biological Chemistry

107

109

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Phosphatidyl-myo-inositol mannosides (PIMs) are unique glycolipids found in abundant quantities in the inner and outer membranes of the cell envelope of all Mycobacterium species. They are based on a phosphatidyl-myo-inositol lipid anchor carrying one to six mannose residues and up to four acyl chains. PIMs are considered not only essential structural components of the cell envelope but also the structural basis of the lipoglycans (lipomannan and lipoarabinomannan), all important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy. Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis and regulation is still incomplete. Recent advances in the identification of key proteins involved in PIM biogenesis and the determination of the three-dimensional structures of the essential phosphatidylmyo-inositol mannosyltransferase PimA and the lipoprotein LpqW have led to important insights into the molecular basis of this pathway.myo-Inositol, as a phospholipid constituent, was first reported in mycobacteria by R. J. Anderson in 1930 (1). Subsequently, the presence of phosphatidyl-myo-inositol (PI) 3 dimannosides (PIM 2 ) and PI pentamannosides (PIM 5 ) was recognized in Mycobacterium tuberculosis (2, 3). Over the past 40 years, the structure of the complete family of PI mannosides (PIM 1 -PIM 6 ) in various Mycobacterium spp. and related Actinomycetes has been defined, first as deacylated glycerophosphoryl-myo-inositol mannosides and later as the fully acylated native molecules (4). PIMs and metabolically related lipoglycans comprising lipomannan (LM) and lipoarabinomannan (LAM) are noncovalently anchored through their PI moiety to the inner and outer membranes of the cell envelope (5, 6) and play various essential although poorly defined roles in mycobacterial physiology. They are also thought to be important virulence factors during the infection cycle of M. tuberculosis. Aided by the availability of a growing number of genome sequences from lipoglycan-producing Actinomycetes, developments in the genetic manipulation of these organisms, and advances in our understanding of the molecular processes underlying sugar transfer in Corynebacterianeae, considerable progress was made over the last 10 years in identifying the enzymes associated with the biogenesis of PIM, LM, and LAM (for recent review, see Refs. 7 and 8). The precise chemical definition of these molecules from various Actinomycetes combined with comparative analyses of their interactions with the host immune system also has shed light on their structure-function relationships (9).In this minireview, we present some key enzymatic, structural, and topological aspects of the biogenesis of PIMs, a pathway that may represent a paradigm for that of other mycobacterial complex (glyco)lipids. The elucidation of this pathway has helped in our understanding of the pathogenesis of tuberculosis and revealed new opportunities for drug discovery. Chem...

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Section: Pim 2 Biosynthesissupporting

confidence: 83%

Molecular Basis of Phosphatidyl-myo-inositol Mannoside Biosynthesis and Regulation in Mycobacteria

Guerin

Korduláková

Alzari

et al. 2010

Journal of Biological Chemistry

107

109

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“…Replacing S192 with alanine affects all kinetic parameters, including the K m for Lipid I (Table 2). MurG utilizes a sequential ordered mechanism in which UDPGlcNAc binds first (23), and it is possible that conformational changes in the GGS loop play a role in the adjustments required for Lipid I binding, as well as contributing directly to UDPGlcNAc binding.…”

Section: Resultsmentioning

confidence: 99%

“…The kinetic parameters of the enzymes were measured by using the continuous fluorescence assay described previously (23). The apparent K m for the donor was determined at a fixed concentration of Lipid I analogue (100 M) because of the limited supply of the synthetic substrate.…”

Section: Methodsmentioning

confidence: 99%

“…This superfamily is also proposed to include O-linked GlcNAc transferase, a GTase that posttranslationally modifies transcription factors and other proteins in eukaryotes (21,22). Despite its evident importance, this superfamily is not as well understood as the GT-A superfamily, in part because there have been no crystal structures of any GT-B enzymes with intact glycosyl donor or acceptor molecules (23). Escherichia coli MurG belongs to the GT-B superfamily, and the x-ray structure of the MurG:UDPGlcNAc complex presented here, combined with genomic analysis of the superfamily, provides information on the origins of substrate selectivity and how it can be manipulated.…”

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confidence: 99%

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Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases

Chen

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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218

236

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MurG is an essential glycosyltransferase that forms the glycosidic linkage between N-acetyl muramyl pentapeptide and N-acetyl glucosamine in the biosynthesis of the bacterial cell wall. This enzyme is a member of a major superfamily of NDP-glycosyltransferases for which no x-ray structures containing intact substrates have been reported. Here we present the 2.5-Å crystal structure of Escherichia coli MurG in complex with its donor substrate, UDPGlcNAc. Combined with genomic analysis of other superfamily members and site-specific mutagenesis of E. coli MurG, this structure sheds light on the molecular basis for both donor and acceptor selectivity for the superfamily. This structural analysis suggests that it will be possible to evolve new glycosyltransferases from prototypical superfamily members by varying two key loops while maintaining the overall architecture of the family and preserving key residues.

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“…PseG (10 l of a 33 M stock solution) was added to each sample and the mixtures were incubated for 2 h at room temperature. The progress of the reactions was monitored using 31 P NMR spectroscopy with integration of the diphosphate signals.…”

Section: Methodsmentioning

confidence: 99%

PseG of Pseudaminic Acid Biosynthesis

Liu

Tanner

2006

Journal of Biological Chemistry

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The flagellin proteins in pathogenic bacteria such as Campylobacter jejuni and Helicobacter pylori are heavily glycosylated with the nine-carbon ␣-keto acid, pseudaminic acid. The presence of this posttranslational modification is absolutely required for assembly of functional flagella. Since motility is required for colonization, pseudaminic acid biosynthesis represents a virulence factor in these bacteria. Pseudaminic acid is generated from UDP-N-acetylglucosamine in five biosynthetic steps. The final step has been shown to involve the condensation of 2,4-diacetamido-2,4,6-trideoxy-L-altrose (6-deoxy-AltdiNAc) with phosphoenolpyruvate as catalyzed by the enzyme pseudaminic acid synthase, NeuB3. The 6-deoxy-AltdiNAc used in this process is generated from its nucleotide-linked form, UDP-6-deoxy-AltdiNAc, by the action of a hydrolase that cleaves the glycosidic bond and releases UDP. This manuscript describes the first characterization of a UDP-6-deoxy-AltdiNAc hydrolase, namely PseG (Cj1312) from C. jejuni. The activity of this enzyme is independent of the presence of divalent metal ions, and the values of the catalytic constants were found to be k cat ‫؍‬ 27 s ؊1 and K m ‫؍‬ 174 M. The enzyme was shown to hydrolyze the substrate with an overall inversion of stereochemistry at C-1 and to utilize a C-O bond cleavage mechanism during catalysis. These results, coupled with homology comparisons, suggest that the closest ancestors to the hydrolase are members of the metal-independent GT-B family of glycosyltransferases that include the enzyme MurG.

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Intrinsic Lipid Preferences and Kinetic Mechanism of Escherichia coli MurG

Cited by 79 publications

References 52 publications

Molecular Basis of Phosphatidyl-myo-inositol Mannoside Biosynthesis and Regulation in Mycobacteria

Molecular Basis of Phosphatidyl-myo-inositol Mannoside Biosynthesis and Regulation in Mycobacteria

Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases

PseG of Pseudaminic Acid Biosynthesis

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