Ucomicrobium is evolutionarily related to the genus Chlamydia [3]. Interestingly, we were able to identify all the genes that are involved in the de novo anabolism of PG from the annotated genome of V. spinosum (Table 1). The MurE ortholog from Chlamydia trachomatis was identified and was shown to be an authentic MurE enzyme, even though PG cannot be detected from the bacterium using methods developed thus far [26]. Unlike C. trachomatis, we were able to isolate and detect PG from V. spinosum in addition to quantifying all the major components of the macromolecule. V. spinosum is an attractive candidate model organism to address questions relating to: i) the chlamydial PG paradox; and ii) the feasibility and plausibility of whether thenewly discovered DapL enzyme is a potential target for antibiotic development given the fact the enzyme is involved in the synthesis of both PG and lysine. Title Loaded From File MurEVs shares 37 and 35 amino acid identity to the MurE orthologs from C. trachomatis and E. coli, respectively. With regards to the substrate specificity of the enzyme, MurEVs resembles that of the C. trachomatis and E. coli orthologs by showing preference for meso-A2pm. The enzyme incorporated very weakly the two other stereoisomers of A2pm; it was unable to incorporate L-lysine and Lornithine, two structurally related diamine compounds. Therefore, MurEVs is highly specific for meso-A2pm.MurE from Verrucomicrobium spinosum DSM 4136TFigure 5. Multiple amino acid sequence alignment of five representative sequences of MurE. The residues that are predicted to be involved in binding in the active site are marked with a star below the sequence. The sequence identity score against MurE from V. spinosum was: C. 1315463 trachomatis, 37 ; E. coli, 35 ; P. carotovorum, 36 ; and M. tuberculosis. The multiple amino acid sequence alignment figure was generated using the ESPript 2.2 server (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). doi:10.1371/journal.pone.0066458.gThe enzyme’s optimum catalytic profile with respect to pH, temperature and [Mg2+] was examined to define optimum assay conditions and also gauge its similarity with other known MurE enzymes. MurEVs displays maximum activity at pH 9.6, which is Title Loaded From File slightly higher than those found in E. coli (pH 8.0?.2) and C. trachomatis (pH 8.0?.6) Mur ligases [15]. The optimal temperature for MurEVs (44?6uC) seems somewhat high but difficult to compare with other orthologs and paralogs since this parameter is almost never mentioned. These unusual values for MurEVs might be attributed to environmental factors such as the natural habitat(s) of the organism. As for the optimal [Mg2+] concentration, it falls within the range (5?00 mM) found for E. coli and C. trachomatis Mur ligases [15,26,30].The maximum velocity of 36 mmol?min21?mg21 for the MurEVs using saturating levels of all substrates is approximately 110, 26 and 14 times more than those of MurECt, MurEEc and MurE from Pseudomonas aeruginosa, respectively [15,26,31]. Whereas the higher specific activity of MurEVs with respect to MurECt can easily be explained by the fact that Chlamydiae are slowgrowing, primarily intracellular organisms [26], we have no explanation for the difference between MurEVs and the orthologs from E. coli and P. aeruginosa. Primary sequence analysis showed that MurEVs contains ten out of the sixteen amino acids that make up the active site of the enzyme including the DNPR motif. The DNPR motif is conserved among MurEs that have been experimen.Ucomicrobium is evolutionarily related to the genus Chlamydia [3]. Interestingly, we were able to identify all the genes that are involved in the de novo anabolism of PG from the annotated genome of V. spinosum (Table 1). The MurE ortholog from Chlamydia trachomatis was identified and was shown to be an authentic MurE enzyme, even though PG cannot be detected from the bacterium using methods developed thus far [26]. Unlike C. trachomatis, we were able to isolate and detect PG from V. spinosum in addition to quantifying all the major components of the macromolecule. V. spinosum is an attractive candidate model organism to address questions relating to: i) the chlamydial PG paradox; and ii) the feasibility and plausibility of whether thenewly discovered DapL enzyme is a potential target for antibiotic development given the fact the enzyme is involved in the synthesis of both PG and lysine. MurEVs shares 37 and 35 amino acid identity to the MurE orthologs from C. trachomatis and E. coli, respectively. With regards to the substrate specificity of the enzyme, MurEVs resembles that of the C. trachomatis and E. coli orthologs by showing preference for meso-A2pm. The enzyme incorporated very weakly the two other stereoisomers of A2pm; it was unable to incorporate L-lysine and Lornithine, two structurally related diamine compounds. Therefore, MurEVs is highly specific for meso-A2pm.MurE from Verrucomicrobium spinosum DSM 4136TFigure 5. Multiple amino acid sequence alignment of five representative sequences of MurE. The residues that are predicted to be involved in binding in the active site are marked with a star below the sequence. The sequence identity score against MurE from V. spinosum was: C. 1315463 trachomatis, 37 ; E. coli, 35 ; P. carotovorum, 36 ; and M. tuberculosis. The multiple amino acid sequence alignment figure was generated using the ESPript 2.2 server (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). doi:10.1371/journal.pone.0066458.gThe enzyme’s optimum catalytic profile with respect to pH, temperature and [Mg2+] was examined to define optimum assay conditions and also gauge its similarity with other known MurE enzymes. MurEVs displays maximum activity at pH 9.6, which is slightly higher than those found in E. coli (pH 8.0?.2) and C. trachomatis (pH 8.0?.6) Mur ligases [15]. The optimal temperature for MurEVs (44?6uC) seems somewhat high but difficult to compare with other orthologs and paralogs since this parameter is almost never mentioned. These unusual values for MurEVs might be attributed to environmental factors such as the natural habitat(s) of the organism. As for the optimal [Mg2+] concentration, it falls within the range (5?00 mM) found for E. coli and C. trachomatis Mur ligases [15,26,30].The maximum velocity of 36 mmol?min21?mg21 for the MurEVs using saturating levels of all substrates is approximately 110, 26 and 14 times more than those of MurECt, MurEEc and MurE from Pseudomonas aeruginosa, respectively [15,26,31]. Whereas the higher specific activity of MurEVs with respect to MurECt can easily be explained by the fact that Chlamydiae are slowgrowing, primarily intracellular organisms [26], we have no explanation for the difference between MurEVs and the orthologs from E. coli and P. aeruginosa. Primary sequence analysis showed that MurEVs contains ten out of the sixteen amino acids that make up the active site of the enzyme including the DNPR motif. The DNPR motif is conserved among MurEs that have been experimen.
http://calcium-channel.com
Calcium Channel
