Tally authenticated. A homology model, based on the well characterized MurEEc enzymeMurE from Verrucomicrobium spinosum DSM 4136TTable 3. Analysis of crude and purified PG from V. spinosum DSM 4136T.ConstituentMolar ratio (Calculated with GlcN = 1.0) Crude PGa Purified PGa 0.04 0.03 0.03 0.80 0.91 0.02 0.06 1.51 0.04 1.10 0 0.03 0.05 0.03 0.03 1.0 0.04 0.02 0.Asp Thr Ser Mur Glu Pro Gly Ala Val A2pm Met Ile Leu Tyr Phe GlcN Lys His Arg1.22 0.62 0.63 0.79 2.33 0.47 0.95 2.47 0.54 1.02 0 0.42 0.86 0.34 0.39 1.0 0.76 0.22 0.a Crude and purified PG designate the macromolecule before and after, respectively, treatment with pancreatin, pronase and trypsin (see Materials and Methods). doi:10.1371/journal.pone.0066458.tbacteriostatic effect, as already observed for other enzymes of the diaminopimelate/L-lysine pathway [33,34]. The genomes of animals and particularly humans do not possess the genetic machinery to facilitate the biosynthesis of diaminopimelate/L-lysine de novo. Therefore, animals must acquire L-lysine through dietary means. Thus there is a unique opportunity to assess the essentiality of enzymes that are important for cell wall and protein synthesis from eubacteria. V. spinosum is an attractive model bacterial system based on the fact that the organism is closely related to Chlamydia, which was found to use the DapL pathway to diaminopimelate/L-lysine. Bioinformatic analysis shows that the sequenced and annotated genomes of bacteria belonging to the genus Chlamydia contain putative dapL orthologs (data not shown). V. spinosum is aerobic and facile to culture using commercially available media Title Loaded From File because it is not an obligate intracellular bacterium as is the case with Chlamydia. Importantly, the bacterium is not pathogenic to mammals based on what we currently know. Since the genome of the organism can be genetically modified using transposon mutagenesis, analysis of genes that are essential for V. spinosum that are involved in the diaminopimelate/L-lysine biosynthesis can be the focus of future studies [10,35]. Here we present the identification and characterization of the first Mur ligase namely, MurE from the bacterium V. spinosum. Bioinformatic and biochemical analyses provide evidence that the bacterium is able to synthesize PG de novo. In vivo analysis shows that MurEVs is an authentic meso-A2pm adding enzyme. This was further validated by in vitro analyses that show that the kinetic and physical Biogenesis such as IsaA.the expression of these genes, except for properties are consistent with MurE orthologs that have been experimentally confirmed. Finally, primary amino acid sequence and structural analysis based on protein modeling show that key amino acids that are involved in substrate binding and or catalysis are conserved in MurEVs.(PDB id: 1E8C), was developed to examine the sequence further and consider the consequences of differences within the MurEVs active site. The MurEVs enzyme is likely to comprise three domains, A, B and C, each of which contribute amino acid residues to the active site. Nearly all of the active site moieties (10 of 16) known to interact with the substrates and products are conserved in the MurEVs active site. Overall, the homology model 23977191 is entirely consistent with our validated function of MurEVs and suggests that the enzyme binds the substrates in a similar way to other known MurE enzymes. Even though the diaminopimelate/L-lysine pathway have been the subject and focus of numerous studies regarding the development of antibiotics, no novel antibiotics have been.Tally authenticated. A homology model, based on the well characterized MurEEc enzymeMurE from Verrucomicrobium spinosum DSM 4136TTable 3. Analysis of crude and purified PG from V. spinosum DSM 4136T.ConstituentMolar ratio (Calculated with GlcN = 1.0) Crude PGa Purified PGa 0.04 0.03 0.03 0.80 0.91 0.02 0.06 1.51 0.04 1.10 0 0.03 0.05 0.03 0.03 1.0 0.04 0.02 0.Asp Thr Ser Mur Glu Pro Gly Ala Val A2pm Met Ile Leu Tyr Phe GlcN Lys His Arg1.22 0.62 0.63 0.79 2.33 0.47 0.95 2.47 0.54 1.02 0 0.42 0.86 0.34 0.39 1.0 0.76 0.22 0.a Crude and purified PG designate the macromolecule before and after, respectively, treatment with pancreatin, pronase and trypsin (see Materials and Methods). doi:10.1371/journal.pone.0066458.tbacteriostatic effect, as already observed for other enzymes of the diaminopimelate/L-lysine pathway [33,34]. The genomes of animals and particularly humans do not possess the genetic machinery to facilitate the biosynthesis of diaminopimelate/L-lysine de novo. Therefore, animals must acquire L-lysine through dietary means. Thus there is a unique opportunity to assess the essentiality of enzymes that are important for cell wall and protein synthesis from eubacteria. V. spinosum is an attractive model bacterial system based on the fact that the organism is closely related to Chlamydia, which was found to use the DapL pathway to diaminopimelate/L-lysine. Bioinformatic analysis shows that the sequenced and annotated genomes of bacteria belonging to the genus Chlamydia contain putative dapL orthologs (data not shown). V. spinosum is aerobic and facile to culture using commercially available media because it is not an obligate intracellular bacterium as is the case with Chlamydia. Importantly, the bacterium is not pathogenic to mammals based on what we currently know. Since the genome of the organism can be genetically modified using transposon mutagenesis, analysis of genes that are essential for V. spinosum that are involved in the diaminopimelate/L-lysine biosynthesis can be the focus of future studies [10,35]. Here we present the identification and characterization of the first Mur ligase namely, MurE from the bacterium V. spinosum. Bioinformatic and biochemical analyses provide evidence that the bacterium is able to synthesize PG de novo. In vivo analysis shows that MurEVs is an authentic meso-A2pm adding enzyme. This was further validated by in vitro analyses that show that the kinetic and physical properties are consistent with MurE orthologs that have been experimentally confirmed. Finally, primary amino acid sequence and structural analysis based on protein modeling show that key amino acids that are involved in substrate binding and or catalysis are conserved in MurEVs.(PDB id: 1E8C), was developed to examine the sequence further and consider the consequences of differences within the MurEVs active site. The MurEVs enzyme is likely to comprise three domains, A, B and C, each of which contribute amino acid residues to the active site. Nearly all of the active site moieties (10 of 16) known to interact with the substrates and products are conserved in the MurEVs active site. Overall, the homology model 23977191 is entirely consistent with our validated function of MurEVs and suggests that the enzyme binds the substrates in a similar way to other known MurE enzymes. Even though the diaminopimelate/L-lysine pathway have been the subject and focus of numerous studies regarding the development of antibiotics, no novel antibiotics have been.
