Substrate Binding Affinity of Pseudomonas aeruginosa Membrane-Bound Lytic Transglycosylase B by Hydrogen−Deuterium Exchange MALDI MS

Lytic transglycosylases cleave the β-(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 identi...

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Veröffentlicht in:Biochemistry (Easton) 2004-09, Vol.43 (35), p.11275-11282
Hauptverfasser: Reid, Christopher W., Brewer, Dyanne, Clarke, Anthony J.
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Brewer, Dyanne
Clarke, Anthony J.
description Lytic transglycosylases cleave the β-(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.
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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. 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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. 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subjects Acetylmuramyl-Alanyl-Isoglutamine - analogs & derivatives
Acetylmuramyl-Alanyl-Isoglutamine - metabolism
Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
Bacteriolysis
Binding Sites
Cell Wall - metabolism
Chromatography, Gel
Deuterium Exchange Measurement - methods
Escherichia coli
Glycosyltransferases - chemistry
Glycosyltransferases - metabolism
Kinetics
Ligands
Muramic Acids - metabolism
Protein Binding
Protein Conformation
Protein Denaturation
Pseudomonas aeruginosa
Pseudomonas aeruginosa - enzymology
Pseudomonas aeruginosa - physiology
Solubility
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
Substrate Specificity
title Substrate Binding Affinity of Pseudomonas aeruginosa Membrane-Bound Lytic Transglycosylase B by Hydrogen−Deuterium Exchange MALDI MS
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