Characterization of soluble and membrane-bound family 3 lytic transglycosylases from Pseudomonas aeruginosa.
Lytic transglycosylases cleave the beta,1-->4 glycosidic linkages between the N-acetylmuramoyl (MurNAc) and N-acetylglucosaminyl (GlcNAc) residues of peptidoglycan with the concomitant formation of 1,6-anhydro-N-acetylmuramyl reaction products. The genes encoding two hypothetical lytic transglycosylases were identified in the genome of Pseudomonas aeruginosa PAO1 by a BLAST search using membrane-bound lytic transglycosylase B (MltB) from Escherichia coli as the query. The two genes were amplified by PCR and cloned as fusion proteins with C-terminal hexa-His sequences. Expression studies of the two genes in E. coli in the presence of [(3)H]palmitate resulted in the labeling of only one of the two enzymes. This enzyme, named MltB, was overexpressed to form insoluble inclusion bodies. Its gene was engineered to produce a truncated form of the enzyme lacking its N-terminal 17 residues which includes Cys17, the putative site of lipidation. This MltB derivative (named sMltB) was shown to not label with [(3)H]palmitate, and it was overexpressed in soluble form. The second, nonlabeled enzyme was overexpressed in soluble form and hence was named soluble lytic transglycosylase B (SltB). Both sMltB and SltB were purified to apparent homogeneity by a combination of affinity (Ni(2+)-NTA), cation-exchange (Mono S), and gel permeation (Superdex 75) chromatographies. The reaction products released by the two enzymes from purified, insoluble peptidoglycan were characterized by a novel high-performance anion-exchange chromatography (HPAEC) assay. Both enzymes produced the same three major soluble products which were identified as anhydromuropeptides based on ESI-MS analysis (cross-linked anhydrodisaccharide-tetrasaccharide, m/z obs 1824.9; anhydrodisaccharide-pentapeptide, m/z obs 922.2; and anhydrodisaccharide-tripeptide, m/z obs 851.3. The Michaelis-Menten kinetic parameters were also determined for the two enzymes using the same insoluble peptidoglycan substrate by aminosugar compositional analysis of soluble reaction products. At pH 5.8 and in the presence of 0.1% Triton, SltB was found to be more catalytically efficient, as reflected by its k(cat)/K(M) value, than sMltB.
Blackburn
Neil T
NT
Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Clarke
Anthony J
AJ
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005838:Genotype; D006027:Glycosides / Q000378:metabolism; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009391:Nephelometry and Turbidimetry; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D006027:Glycosides; D011994:Recombinant Proteins; D016695:Glycosyltransferases; C021815:murein transglycosylase