Structure and activity of ChiX, a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens

Richard A. Owen, Paul K. Fyfe, Adam Lodge, Jacob Biboy, Waldemar Vollmer, William N. Hunter, Frank Sargent (Lead / Corresponding author)

Research output: Contribution to journalArticle

4 Citations (Scopus)
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Abstract

The Gram‐negative bacterium Serratia marcescens secretes a number of proteins that are involved in extracellular chitin degradation. This so‐called chitinolytic machinery includes three types of chitinase enzymes and a lytic polysaccharide monooxygenase. An operon has been identified in S. marcescens, chiWXYZ, that isthought to be involved in the secretion of the chitinolytic machinery. Genetic evidence points to the ChiX protein being a key player in the secretion mechanism, since deletion of the chiX gene in S. marcescens led to a mutant strain blocked for secretion of all members of the chitinolytic machinery. In this work, a detailed structural and biochemical characterisation of ChiX is presented. The high resolution crystal structure of ChiX reveals the protein to be a member of the LAS family of peptidases. ChiX is shown to be a Zinc‐containing metalloenzyme and in vitro assays demonstrate ChiX is an L‐Ala D‐Glu endopeptidase that cleaves the crosslinks in bacterial peptidoglycan. This catalytic activity is shown to be intimately linked with the secretion of the chitinolytic machinery, since substitution of the ChiX Asp‐120 residue results in a variant protein that is both unable to digest peptidoglycan and cannot rescue the phenoytype of a chiX mutant strain.
Original languageEnglish
Pages (from-to)415-428
Number of pages14
JournalBiochemical Journal
Volume475
Issue number2
Early online date11 Dec 2017
DOIs
Publication statusPublished - 23 Jan 2018

Keywords

  • Bacterial chitinase secretion
  • Peptidoglycan hydrolase
  • Crystal structure
  • Zinc enzyme
  • Anomalous dispersion
  • Mutagenesis  
  • Aspartic Acid/chemistry
  • Substrate Specificity
  • Crystallography, X-Ray
  • Recombinant Proteins/chemistry
  • Cloning, Molecular
  • Bacterial Proteins/chemistry
  • Protein Interaction Domains and Motifs
  • Chitinases/genetics
  • Binding Sites
  • Genetic Vectors/chemistry
  • Protein Conformation, alpha-Helical
  • Gene Expression
  • N-Acetylmuramoyl-L-alanine Amidase/chemistry
  • Operon
  • Zinc/chemistry
  • Models, Molecular
  • Escherichia coli/genetics
  • Serratia marcescens/enzymology
  • Amino Acid Motifs
  • Hydrolysis
  • Peptidoglycan/chemistry
  • Protein Conformation, beta-Strand
  • Protein Binding
  • Chitin/metabolism
  • Gene Expression Regulation, Bacterial

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