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. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (or dl-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-terminal l-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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Joint Center for Structural Genomics‡ Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California, USA.
authors.forename: Qingping
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris Sud, Orsay, France mengin-lecreulx@u-psud.fr wilson@scripps.edu.
authors.forename: Dominique
authors.initials: D
authors.lastname: Mengin-Lecreulx
authors.affiliation: Joint Center for Structural Genomics‡ Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Xueqian W
authors.initials: XW
authors.lastname: Liu
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris Sud, Orsay, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Patin
authors.affiliation: Joint Center for Structural Genomics‡ Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, California, USA.
authors.forename: Carol L
authors.initials: CL
authors.lastname: Farr
authors.affiliation: Joint Center for Structural Genomics‡ Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, California, USA.
authors.forename: Joanna C
authors.initials: JC
authors.lastname: Grant
authors.affiliation: Joint Center for Structural Genomics‡ Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California, USA.
authors.forename: Hsiu-Ju
authors.initials: HJ
authors.lastname: Chiu
authors.affiliation: Joint Center for Structural Genomics‡ Center for Research in Biological Systems, University of California San Diego, La Jolla, California, USA Program on Bioinformatics and Systems Biology, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA.
authors.forename: Lukasz
authors.initials: L
authors.lastname: Jaroszewski
authors.affiliation: Joint Center for Structural Genomics‡ Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, California, USA.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Knuth
authors.affiliation: Joint Center for Structural Genomics‡ Center for Research in Biological Systems, University of California San Diego, La Jolla, California, USA Program on Bioinformatics and Systems Biology, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Godzik
authors.affiliation: Joint Center for Structural Genomics‡ Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, California, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lesley
authors.affiliation: Joint Center for Structural Genomics‡ Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Marc-André
authors.initials: MA
authors.lastname: Elsliger
authors.affiliation: Joint Center for Structural Genomics‡ Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California, USA.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Deacon
authors.affiliation: Joint Center for Structural Genomics‡ Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA mengin-lecreulx@u-psud.fr wilson@scripps.edu.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Wilson
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D018909:src Homology Domains
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; D000626:Aminopeptidases; C075408:cysteine aminopeptidase