Substrate binding affinity of Pseudomonas aeruginosa membrane-bound lytic transglycosylase B by hydrogen-deuterium exchange MALDI MS.
Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer, peptidoglycan, between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. With 72% amino acid sequence identity between the enzymes, the theoretical structure of the membrane-bound lytic transglycosylase B (MltB) from Psuedomonas aeruginosa was modeled on the known crystal structure of Escherichia coli Slt35, the soluble derivative of its MltB. Of the twelve residues in Slt35 known to make contacts with peptidoglycan derivatives in Slt35, nine exist in the same position in the P. aeruginosa homologue, with two others only slightly displaced. To probe the binding properties of an engineered soluble form of the P. aeruginosa MltB, a SUPREX method involving hydrogen/deuterium exchange coupled with MALDI mass spectrometry detection was developed. Dissociation constants were calculated for a series of peptidoglycan components and compared to those obtained by difference UV absorption spectroscopy. These data indicated that GlcNAc alone does not bind to MltB with any measurable affinity but it does contribute to the binding of GlcNAc-MurNAc-dipeptide. With the MurNAc series of ligands, significant binding contributions are made through both the N-acetyl and C-3 lactyl moieties of the aminosugar with additional contributions to binding provided by associated peptides.
Reid
Christopher W
CW
Guelph-Waterloo Center for Graduate Work in Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
Brewer
Dyanne
D
Clarke
Anthony J
AJ
D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000031:analogs & derivatives* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001433:Bacteriolysis; D001665:Binding Sites; D002473:Cell Wall / Q000378:metabolism; D002850:Chromatography, Gel; D041961:Deuterium Exchange Measurement / Q000379:methods*; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009112:Muramic Acids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000502:physiology; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D008024:Ligands; D009112:Muramic Acids; C031651:N-acetylmuramic acid; D000119:Acetylmuramyl-Alanyl-Isoglutamine; C023868:N-acetyl-beta-glucosaminyl-N-acetylmuramyl-alanylisoglutamine; D016695:Glycosyltransferases; C021815:murein transglycosylase