skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

Abstract

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer thatmore » envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Authors:
 [1];  [2];  [3];  [2];  [4];  [5];  [1];  [6];  [5];  [6];  [4];  [3];  [1];  [3]
  1. SLAC National Accelerator Lab., Menlo Park, CA (United States)
  2. Univ. Paris Sud, Orsay (France)
  3. SLAC National Accelerator Lab., Menlo Park, CA (United States); The Scripps Research Institute, La Jolla, CA (United States)
  4. SLAC National Accelerator Lab., Menlo Park, CA (United States); The Scripps Research Institute, La Jolla, CA (United States); Genomics Institute of the Novartis Research Foundation, San Diego, CA (United States)
  5. SLAC National Accelerator Lab., Menlo Park, CA (United States); Univ. of California San Diego, La Jolla, CA (United States)
  6. SLAC National Accelerator Lab., Menlo Park, CA (United States); Univ. of California San Diego, La Jolla, CA (United States); Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA (United States)
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1233189
Grant/Contract Number:  
AC03-76SF00515; AC02-76SF00515
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
mBio (Online)
Additional Journal Information:
Journal Volume: 6; Journal Issue: 5; Journal ID: ISSN 2150-7511
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Xu, Qingping, Mengin-Lecreulx, Dominique, Liu, Xueqian W., Patin, Delphine, Farr, Carol L., Grant, Joanna C., Chiu, Hsiu -Ju, Jaroszewski, Lukasz, Knuth, Mark W., Godzik, Adam, Lesley, Scott A., Elsliger, Marc -André, Deacon, Ashley M., and Wilson, Ian A. Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains. United States: N. p., 2015. Web. doi:10.1128/mBio.02327-14.
Xu, Qingping, Mengin-Lecreulx, Dominique, Liu, Xueqian W., Patin, Delphine, Farr, Carol L., Grant, Joanna C., Chiu, Hsiu -Ju, Jaroszewski, Lukasz, Knuth, Mark W., Godzik, Adam, Lesley, Scott A., Elsliger, Marc -André, Deacon, Ashley M., & Wilson, Ian A. Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains. United States. https://doi.org/10.1128/mBio.02327-14
Xu, Qingping, Mengin-Lecreulx, Dominique, Liu, Xueqian W., Patin, Delphine, Farr, Carol L., Grant, Joanna C., Chiu, Hsiu -Ju, Jaroszewski, Lukasz, Knuth, Mark W., Godzik, Adam, Lesley, Scott A., Elsliger, Marc -André, Deacon, Ashley M., and Wilson, Ian A. 2015. "Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains". United States. https://doi.org/10.1128/mBio.02327-14. https://www.osti.gov/servlets/purl/1233189.
@article{osti_1233189,
title = {Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains},
author = {Xu, Qingping and Mengin-Lecreulx, Dominique and Liu, Xueqian W. and Patin, Delphine and Farr, Carol L. and Grant, Joanna C. and Chiu, Hsiu -Ju and Jaroszewski, Lukasz and Knuth, Mark W. and Godzik, Adam and Lesley, Scott A. and Elsliger, Marc -André and Deacon, Ashley M. and Wilson, Ian A.},
abstractNote = {Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.},
doi = {10.1128/mBio.02327-14},
url = {https://www.osti.gov/biblio/1233189}, journal = {mBio (Online)},
issn = {2150-7511},
number = 5,
volume = 6,
place = {United States},
year = {Tue Sep 15 00:00:00 EDT 2015},
month = {Tue Sep 15 00:00:00 EDT 2015}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 28 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Peptidoglycan turnover and recycling in Gram-positive bacteria
journal, July 2011


Overview of the CCP 4 suite and current developments
journal, March 2011


Structural Basis of Murein Peptide Specificity of a γ-D-Glutamyl-L-Diamino Acid Endopeptidase
journal, February 2009


Structure and Function of a Novel LD-Carboxypeptidase A Involved in Peptidoglycan Recycling
journal, October 2013


Distributed structure determination at the JCSG
journal, March 2011


Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts
journal, January 2008


Recycling of murein by Escherichia coli.
journal, January 1985


Identification and Characterization of Novel Cell Wall Hydrolase CwlT: A TWO-DOMAIN AUTOLYSIN EXHIBITING N-ACETYLMURAMIDASE AND dl-ENDOPEPTIDASE ACTIVITIES
journal, February 2008


Refinement of severely incomplete structures with maximum likelihood in BUSTER–TNT
journal, November 2004


SignalP 4.0: discriminating signal peptides from transmembrane regions
journal, September 2011


An automated system to mount cryo-cooled protein crystals on a synchrotron beamline, using compact sample cassettes and a small-scale robot
journal, November 2002


Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline
journal, August 2002


Bacterial peptidoglycan (murein) hydrolases
journal, March 2008


Atomic Structures of the Human Immunophilin FKBP-12 Complexes with FK506 and Rapamycin
journal, January 1993


Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0
journal, October 2003


How Bacteria Consume Their Own Exoskeletons (Turnover and Recycling of Cell Wall Peptidoglycan)
journal, June 2008


SH3 domains in prokaryotes
journal, April 1999


XDS
journal, January 2010


Bacterial cell-wall recycling: Bacterial cell-wall recycling
journal, November 2012


Highly Sensitive Detection of Individual HEAT and ARM Repeats with HHpred and COACH
journal, September 2009


Reverse-phase high-pressure liquid chromatography of uridine diphosphate N-acetylmuramyl peptide precursors of bacterial cell wall peptidoglycan
journal, June 1981


JLigand : a graphical tool for the CCP 4 template-restraint library
journal, March 2012


Peptidoglycan Molecular Requirements Allowing Detection by the Drosophila Immune Deficiency Pathway
journal, December 2004


The JCSG high-throughput structural biology pipeline
journal, September 2010


Breaching the great wall: peptidoglycan and microbial interactions
journal, August 2006


Substrate-Induced Inactivation of the Escherichia coli AmiD N-Acetylmuramoyl-l-Alanine Amidase Highlights a New Strategy To Inhibit This Class of Enzyme
journal, February 2009


Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens
journal, March 2010


A short history of SHELX
journal, December 2007


MolProbity : all-atom structure validation for macromolecular crystallography
journal, December 2009


Coot model-building tools for molecular graphics
journal, November 2004


REFMAC 5 for the refinement of macromolecular crystal structures
journal, March 2011


Matt: Local Flexibility Aids Protein Multiple Structure Alignment
journal, January 2008


Classical electrostatics in biology and chemistry
journal, May 1995


Structure of the γ- D -glutamyl- L -diamino acid endopeptidase YkfC from Bacillus cereus in complex with L -Ala-γ- D -Glu: insights into substrate recognition by NlpC/P60 cysteine peptidases
journal, July 2010


Peptidoglycan structure and architecture
journal, March 2008


An approach to rapid protein crystallization using nanodroplets
journal, March 2002


On the active site of proteases. III. Mapping the active site of papain; specific peptide inhibitors of papain
journal, September 1968


Structure-Guided Functional Characterization of DUF1460 Reveals a Highly Specific NlpC/P60 Amidase Family
journal, December 2014


Structures of a Bifunctional Cell Wall Hydrolase CwlT Containing a Novel Bacterial Lysozyme and an NlpC/P60 dl-Endopeptidase
journal, January 2014


Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7
journal, June 2008


Homology models guide discovery of diverse enzyme specificities among dipeptide epimerases in the enolase superfamily
journal, March 2012


The Drosophila immune system detects bacteria through specific peptidoglycan recognition
journal, April 2003


Peptidoglycan Hydrolases of Escherichia coli
journal, November 2011


Protein modules and signalling networks
journal, February 1995


Works referencing / citing this record:

Porphyromonas gingivalis genes conferring fitness in a tobacco‐rich environment
journal, December 2019


Enterococcus faecium secreted antigen A generates muropeptides to enhance host immunity and limit bacterial pathogenesis.
text, January 2019