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

Richard A Owen,William N Hunter,Adam Lodge,Frank Sargent,Waldemar Vollmer,Paul K Fyfe,Jacob Biboy

doi:10.1042/bcj20170633

Richard A Owen, William N Hunter + Show 5 more

Open Access

https://doi.org/10.1042/bcj20170633

Copy DOI

Journal: Biochemical Journal	Publication Date: Jan 23, 2018
Citations: 14	License type: CC BY 4.0

Affiliation: University of Dundee, Newcastle University

Abstract

The Gram-negative bacterium Serratia marcescens secretes many 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 is thought 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 that ChiX is an l-Ala d-Glu endopeptidase that cleaves the cross-links 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.

Highlights

Serratia marcescens is a Gram-negative opportunistic bacterial pathogen of the Enterobacteriaceae and is estimated to be responsible for ∼1.4% of nosocomial infections [1,2]
In vitro assays establish ChiX as an L-Ala D-Glu endopeptidase that cleaves the cross-links in bacterial peptidoglycan, and sitedirected mutagenesis is used to demonstrate that the chitinase secretion controlled by ChiX is directly attributable to this catalytic activity
Peptidoglycan-degrading enzymes are diverse in structure, mechanism and physiological role and can be found encoded within the genomes of bacteriophage, prokaryotes and eukaryotes [30]

Summary

Introduction

Serratia marcescens is a Gram-negative opportunistic bacterial pathogen of the Enterobacteriaceae and is estimated to be responsible for ∼1.4% of nosocomial infections [1,2]. Inactivation of either chiW or chiX impairs secretion of the entire chitinolytic machinery with the proteins accumulating in the periplasm unable to cross the outer membrane [11]. ChiW is predicted to be an inner membrane protein with three transmembrane domains, whereas ChiX is predicted to function in the periplasm, but does not itself contain an obvious signal peptide. The chitinase-secretion-defective phenotype of a chiW mutant can be rescued if the signal peptide from another protein is attached to ChiX [11]. This suggests that an important role of ChiW is in targeting ChiX to the periplasm of S. marcescens and that the localisation and biochemical function of ChiX is central to the secretion process. In vitro assays establish ChiX as an L-Ala D-Glu endopeptidase that cleaves the cross-links in bacterial peptidoglycan, and sitedirected mutagenesis is used to demonstrate that the chitinase secretion controlled by ChiX is directly attributable to this catalytic activity

Experimental procedures

Results

Discussion