Inetic constants, the same assay was used with various concentrations of one substrate and fixed concentrations of the others. In all cases, the enzyme concentration was chosen so that substrate consumption was ,20 , the linearity being ensured within this interval even at the lowest substrate concentration. Data were fitted to the equation v = VmaxS/(Km+S) by the Levenberg-Marquardt method [19], where v is the initial velocity and S is the substrate concentration, and values 6 standard deviation at 95 of confidence were calculated. The MDFitt software developed by M. Desmadril (UMR 8619, CNRS, Orsay, France) was used for this purpose.Protein expression and purification of the recombinant MurEvsThe E. coli BL21-CodonPlusH (DE3)-RIPL (Agilent Technologies, USA) strain was transformed with the plasmid pET100D::murEVs and grown in LB broth containing 50 mg?mL21 ampicillin and 34 mg?mL21 chloramphenicol at 37uC to an OD600 16574785 of 0.5. Protein expression was induced in 1 L of culture using isopropyl bD-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM for 8 h at 20uC. The cell pellet was lysed by sonication in a buffer consisting of 50 mM sodium phosphate, pH 8.0, and 300 mM NaCl. The Title Loaded From File soluble extract was incubated with 1 mL bed volume of TALON Metal Affinity Resin (Clontech, Mountain View CA, USA) for 30 min at 4uC. The resin was washed 5 times with 30 mL of sonication buffer containing 10 mM imidazole for 15 min each. The enzyme was eluted with 10 mL of sonication buffer containing 250 mM imidazole. The hexa-histidine tag was not removed after protein purification. The pure protein was concentrated in an Amicon Ultra 10,000 molecular weight cutoff filter unit replacing the elution buffer with 20 mM potassium phosphate, pH 7.2, 1 mM dithiothreitol (DTT), 1 mM EDTA and 10 (v/v) glycerol. The protein concentration was determined by quantitative amino acid analysis as described below.Sequence alignment and homology modelingA multiple amino acid sequence alignment between the Mur ligase enzymes of V. spinosum (ZP_02928794.1), Mycobacterium tuberculosis (CCE37632.1), E. coli (NP_414627.1) Chlamydia trachomatis (NP_219774.1) and Pectobacterium carotovorum (ZP_03831119.1) was generated using ClustalW2 (http://www.ebi.ac.uk/Tools/ msa/clustalw2/) with the Gonnet scoring matrix. The homology model of the MurEVs protein was generated using the SWISS-MODEL Protein Modeling Server [20,21,22] (http://swissmodel.expasy.org/) using the E. coli MurE structure as a template PDB id: 1E8C [23], which was identified using a PSI-BLAST search of the MurEVs protein sequence against proteins in the Protein Data Bank using the web server: (http:// blast.ncbi.nlm.nih.gov/). The model was examined by hand for clashes and appropriate geometry using the visualization software PyMOL (The PyMOL Molecular Graphics System, Schrodinger, ?LLC).Purification and analysis of V. spinosum PGPG was prepared and analyzed essentially according to MenginLecreulx et al. [24]. Cells from 1 L of culture were harvested at 4uC and resuspended in 4 (w/v) sodium dodecyl sulfate (SDS) (10 mL?g21 of cell wet weight) under constant and vigorous stirring at 100uC for 30 min. The suspension was incubated overnight at 25uC followed by centrifugation for 1 h at 17,000 rpm. The pellet containing crude PG was washed 5 times with 10 mL of sterile water and stored in water for further analysis. Half of the preparation was used to obtain purified PG. Briefly, the Title Loaded From File following treatments at 3.Inetic constants, the same assay was used with various concentrations of one substrate and fixed concentrations of the others. In all cases, the enzyme concentration was chosen so that substrate consumption was ,20 , the linearity being ensured within this interval even at the lowest substrate concentration. Data were fitted to the equation v = VmaxS/(Km+S) by the Levenberg-Marquardt method [19], where v is the initial velocity and S is the substrate concentration, and values 6 standard deviation at 95 of confidence were calculated. The MDFitt software developed by M. Desmadril (UMR 8619, CNRS, Orsay, France) was used for this purpose.Protein expression and purification of the recombinant MurEvsThe E. coli BL21-CodonPlusH (DE3)-RIPL (Agilent Technologies, USA) strain was transformed with the plasmid pET100D::murEVs and grown in LB broth containing 50 mg?mL21 ampicillin and 34 mg?mL21 chloramphenicol at 37uC to an OD600 16574785 of 0.5. Protein expression was induced in 1 L of culture using isopropyl bD-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM for 8 h at 20uC. The cell pellet was lysed by sonication in a buffer consisting of 50 mM sodium phosphate, pH 8.0, and 300 mM NaCl. The soluble extract was incubated with 1 mL bed volume of TALON Metal Affinity Resin (Clontech, Mountain View CA, USA) for 30 min at 4uC. The resin was washed 5 times with 30 mL of sonication buffer containing 10 mM imidazole for 15 min each. The enzyme was eluted with 10 mL of sonication buffer containing 250 mM imidazole. The hexa-histidine tag was not removed after protein purification. The pure protein was concentrated in an Amicon Ultra 10,000 molecular weight cutoff filter unit replacing the elution buffer with 20 mM potassium phosphate, pH 7.2, 1 mM dithiothreitol (DTT), 1 mM EDTA and 10 (v/v) glycerol. The protein concentration was determined by quantitative amino acid analysis as described below.Sequence alignment and homology modelingA multiple amino acid sequence alignment between the Mur ligase enzymes of V. spinosum (ZP_02928794.1), Mycobacterium tuberculosis (CCE37632.1), E. coli (NP_414627.1) Chlamydia trachomatis (NP_219774.1) and Pectobacterium carotovorum (ZP_03831119.1) was generated using ClustalW2 (http://www.ebi.ac.uk/Tools/ msa/clustalw2/) with the Gonnet scoring matrix. The homology model of the MurEVs protein was generated using the SWISS-MODEL Protein Modeling Server [20,21,22] (http://swissmodel.expasy.org/) using the E. coli MurE structure as a template PDB id: 1E8C [23], which was identified using a PSI-BLAST search of the MurEVs protein sequence against proteins in the Protein Data Bank using the web server: (http:// blast.ncbi.nlm.nih.gov/). The model was examined by hand for clashes and appropriate geometry using the visualization software PyMOL (The PyMOL Molecular Graphics System, Schrodinger, ?LLC).Purification and analysis of V. spinosum PGPG was prepared and analyzed essentially according to MenginLecreulx et al. [24]. Cells from 1 L of culture were harvested at 4uC and resuspended in 4 (w/v) sodium dodecyl sulfate (SDS) (10 mL?g21 of cell wet weight) under constant and vigorous stirring at 100uC for 30 min. The suspension was incubated overnight at 25uC followed by centrifugation for 1 h at 17,000 rpm. The pellet containing crude PG was washed 5 times with 10 mL of sterile water and stored in water for further analysis. Half of the preparation was used to obtain purified PG. Briefly, the following treatments at 3.
