Biochemical characterization of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase (MurE) from Verrucomicrobium spinosum DSM 4136(T.)

PLoS One. 2013 Jun 13;8(6):e66458. doi: 10.1371/journal.pone.0066458. Print 2013.


Verrucomicrobium spinosum is a Gram-negative bacterium that is related to bacteria from the genus Chlamydia. The bacterium is pathogenic towards Drosophila melanogaster and Caenorhabditis elegans, using a type III secretion system to facilitate pathogenicity. V. spinosum employs the recently discovered l,l-diaminopimelate aminotransferase biosynthetic pathway to generate the bacterial cell wall and protein precursors diaminopimelate and lysine. A survey of the V. spinosum genome provides evidence that the bacterium should be able to synthesize peptidoglycan de novo, since all of the necessary genes are present. The enzyme UDP-N-acetylmuramoyl-l-alanyl-d-glutamate: meso-2,6-diaminopimelate ligase (MurE) (E.C. catalyzes a reaction in the cytoplasmic step of peptidoglycan biosynthesis by adding the third amino acid residue to the peptide stem. The murE ortholog from V. spinosum (murE Vs) was cloned and was shown to possess UDP-MurNAc-l-Ala-d-Glu:meso-2,6-diaminopimelate ligase activity in vivo using functional complementation. In vitro analysis using the purified recombinant enzyme demonstrated that MurEVs has a pH optimum of 9.6 and a magnesium optimum of 30 mM. meso-Diaminopimelate was the preferred substrate with a K m of 17 µM, when compared to other substrates that are structurally related. Sequence alignment and structural analysis using homology modeling suggest that key residues that make up the active site of the enzyme are conserved in MurEVs. Our kinetic analysis and structural model of MurEVs is consistent with other MurE enzymes from Gram-negative bacteria that have been characterized. To verify that V. spinosum incorporates diaminopimelate into its cell wall, we purified peptidoglycan from a V. spinosum culture; analysis revealed the presence of diaminopimelate, consistent with that of a bona fide peptidoglycan from Gram-negative bacteria.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism*
  • Gene Expression
  • Genome, Bacterial
  • Glutamic Acid / metabolism*
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Open Reading Frames
  • Peptide Synthases / chemistry
  • Peptide Synthases / genetics
  • Peptide Synthases / isolation & purification
  • Peptide Synthases / metabolism*
  • Peptidoglycan / chemistry
  • Peptidoglycan / metabolism
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Verrucomicrobia / enzymology*
  • Verrucomicrobia / genetics
  • Verrucomicrobia / ultrastructure


  • Bacterial Proteins
  • Peptidoglycan
  • Recombinant Proteins
  • Glutamic Acid
  • Peptide Synthases

Grants and funding

This research was supported by a National Science Foundation (NSF) (MCB-1120541) and a Rochester Institute of Technology (RIT) College of Science 2012 Dean's Research Initiation Grant to AOH. MAS acknowledges a RIT College of Science Faculty Development (FEAD-2012) grant. SEM and DTP were supported by the NSF MCB-1120541 awarded to AOH as undergraduate students in the Bioinformatics program in the Thomas H. Gosnell School of Life Sciences and the Biochemistry program in the School of Chemical and Material Sciences at RIT. RCJD acknowledges: 1) the C.R. Roper Bequest for Fellowship support; 2) the New Zealand Royal Society Marsden Fund for funding support, in part (contract UOC1013); and 3) the U.S. Army Research Laboratory and U.S. Army Research Office under contract/grant number W911NF-11-1-0481 for supported, in part. DP and DB were supported by grants from the Centre National de la Recherche Scientifique (UMR 8619). HS acknowledges the Royal Society of New Zealand and the Japan Society for the Promotion of Science for salary support by the FY 2012 Researcher Exchange Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.