title: Biochemical characterization of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase (MurE) from Verrucomicrobium spinosum DSM 4136(T.).
abstract: 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. 6.3.2.15) 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.
authors.affiliation: The Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, New York, United States of America.
authors.forename: Sean E
authors.initials: SE
authors.lastname: McGroty
authors.forename: Dhivya T
authors.initials: DT
authors.lastname: Pattaniyil
authors.forename: Delphine
authors.initials: D
authors.lastname: Patin
authors.forename: Didier
authors.initials: D
authors.lastname: Blanot
authors.forename: Arvind C
authors.initials: AC
authors.lastname: Ravichandran
authors.forename: Hironori
authors.initials: H
authors.lastname: Suzuki
authors.forename: Renwick C J
authors.initials: RC
authors.lastname: Dobson
authors.forename: Michael A
authors.initials: MA
authors.lastname: Savka
authors.forename: André O
authors.initials: AO
authors.lastname: Hudson
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D016680:Genome, Bacterial; D018698:Glutamic Acid / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D061327:Verrucomicrobia / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure
chemical_list: D001426:Bacterial Proteins; D010457:Peptidoglycan; D011994:Recombinant Proteins; D018698:Glutamic Acid; D010453:Peptide Synthases