Biochemical analysis of NlpC/p60 peptidoglycan hydrolase activity

Byungchul Kim; Juliel Espinosa; Howard C Hang

doi:10.1016/bs.mie.2020.02.017

Biochemical analysis of NlpC/p60 peptidoglycan hydrolase activity

Methods Enzymol. 2020:638:109-127. doi: 10.1016/bs.mie.2020.02.017. Epub 2020 Mar 31.

Authors

Byungchul Kim¹, Juliel Espinosa¹, Howard C Hang²

Affiliations

¹ Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY, United States.
² Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY, United States. Electronic address: hhang@rockefeller.edu.

PMID: 32416909
DOI: 10.1016/bs.mie.2020.02.017

Abstract

The NlpC/p60-family of peptidoglycan hydrolases are key enzymes that facilitate bacterial cell division and also modulate microbe-host interactions. These endopeptidases utilize conserved Cys-His residues in their active site and are expressed in most bacterial species as well as some eukaryotes. Here we describe methods for biochemical analysis of Enterococcus faecium SagA-NlpC/p60 peptidoglycan hydrolase activity (Kim et al., 2019; Rangan et al., 2016), which includes recombinant protein preparation and biochemical analysis using both gel-based and LC-MS profiling of peptidoglycan fragments. These protocols should also facilitate the biochemical analysis of other NlpC/p60 peptidoglycan hydrolases.

Keywords: Enterococcus faecium; NlpC/p60; Peptidoglycan hydrolase; SagA.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Bacterial Proteins / genetics
Catalytic Domain
Cell Wall / metabolism
Crystallography, X-Ray
N-Acetylmuramoyl-L-alanine Amidase* / genetics
N-Acetylmuramoyl-L-alanine Amidase* / metabolism
Peptidoglycan*

Substances

Bacterial Proteins
Peptidoglycan
N-Acetylmuramoyl-L-alanine Amidase

Grants and funding

R01 GM103593/GM/NIGMS NIH HHS/United States