Biochemical Characterisation of Phage Pseudomurein Endoisopeptidases PeiW and PeiP Using Synthetic Peptides.

Ron S Ronimus,Debjit Dey,Amy K Beattie,Catherine M Tootill,Linley R Schofield

doi:10.1155/2015/828693

Abstract

Pseudomurein endoisopeptidases cause lysis of the cell walls of methanogens by cleaving the isopeptide bond Ala-ε-Lys in the peptide chain of pseudomurein. PeiW and PeiP are two thermostable pseudomurein endoisopeptidases encoded by phage ΨM100 of Methanothermobacter wolfei and phages ΨM1 and ΨM2 of Methanothermobacter marburgensis, respectively. A continuous assay using synthetic peptide substrates was developed and used in the biochemical characterisation of recombinant PeiW and PeiP. The advantages of these synthetic peptide substrates over natural substrates are sensitivity, high purity, and characterisation and the fact that they are more easily obtained than natural substrates. In the presence of a reducing agent, purified PeiW and PeiP each showed similar activity under aerobic and anaerobic conditions. Both enzymes required a divalent metal for activity and showed greater thermostability in the presence of Ca2+. PeiW and PeiP involve a cysteine residue in catalysis and have a monomeric native conformation. The kinetic parameters, K M and k cat, were determined, and the ε-isopeptide bond between alanine and lysine was confirmed as the bond lysed by these enzymes in pseudomurein. The new assay may have wider applications for the general study of peptidases and the identification of specific methanogens susceptible to lysis by specific pseudomurein endoisopeptidases.

Highlights

Pseudomurein is one of the many different cell wall polymers present in Archaea, being found only in Methanobacteriales and the genus Methanopyrus [1,2,3,4]
PeiW is encoded by the defective prophage ΨM100 of the thermophile Methanothermobacter wolfei [12], and PeiP is encoded by phage ΨM1 and deletion mutant ΨM2 of the thermophile Methanothermobacter marburgensis [13, 14]
This work describes the development of a continuous synthetic peptide assay which could be used at elevated temperatures and the expression, purification, and biochemical characterisation of recombinant PeiW and PeiP

Summary

Introduction

Pseudomurein is one of the many different cell wall polymers present in Archaea, being found only in Methanobacteriales and the genus Methanopyrus [1,2,3,4]. The main chemical differences in the methanogen pseudomurein cell walls (Figure 1) are (i) the presence of an archaeal-specific sugar, Nacetyltalosaminuronic acid in the glycan backbone (instead of the bacterial N-acetylmuramic acid) that is linked to either N-acetylglucosamine or N-acetylgalactosamine (rather than just N-acetylglucosamine), (ii) the use of β (1-3) bonds in the glycan chain, instead of β (1-4) bonds, (iii) the lack of damino acids in the peptide chain (instead of a mixture of d-, l-, and meso-amino acids), and (iv) the use of ε- and γ-isopeptide bonds in both the peptide and interpeptidyl cross link [3, 5,6,7]. Φmru from Mbb. ruminantium encodes a lytic enzyme, endoisopeptidase PeiR, which was shown to lyse Mbb. ruminantium cells in pure culture but shows little homology to PeiW and PeiP [15]

Methods

Results

Conclusion