Structure-function relationships underlying the dual N-acetylmuramic and N-acetylglucosamine specificities of the bacterial peptidoglycan deacetylase PdaC

Abstract

Bacillus subtilis PdaC (BsPdaC) is a membrane-bound, multidomain peptidoglycan N-deacetylase acting on N-acetylmuramic acid (MurNAc) residues and conferring lysozyme resistance to modified cell wall peptidoglycans. BsPdaC contains a C-terminal family 4 carbohydrate esterase (CE4) catalytic domain, but unlike other MurNAc deacetylases, BsPdaC also has GlcNAc deacetylase activity on chitooligosaccharides (COSs), characteristic of chitin deacetylases. To uncover the molecular basis of this dual activity, here we determined the X-ray structure of the BsPdaC CE4 domain at 1.54 Å resolution and analyzed its mode of action on COS substrates. We found that the minimal substrate is GlcNAc 3 and that activity increases with the degree of glycan polymerization. COS deacetylation kinetics revealed that BsPdaC operates by a multiple-chain mechanism starting at the internal GlcNAc units and leading to deacetylation of all but the reducing-end GlcNAc residues. Interestingly, BsPdaC shares higher sequence similarity with the peptidoglycan GlcNAc deacetylase SpPgdaA than with other MurNAc deacetylases. Therefore, we used liganddocking simulations to analyze the dual GlcNAc-and MurNAcbinding specificities of BsPdaC and compared them with those of SpPgdA and BsPdaA, representing peptidoglycan deacetylases highly specific for GlcNAc or MurNAc residues, respectively. BsP-daC retains the conserved Asp-His-His metal-binding triad characteristic of CE4 enzymes acting on GlcNAc residues, differing from MurNAc deacetylases that lack the metal-coordinating Asp residue. BsPdaC contains short loops similar to those in SpPgdA, resulting in an open binding cleft that can accommodate polymeric substrates. We propose that PdaC is the first member of a new subclass of peptidoglycan MurNAc deacetylases.

In silico and in vitro analysis of an Aspergillus niger chitin deacetylase to decipher its subsite sugar preferences

“…We have performed a more in depth in vitro and in silico analysis on AngCDA than previously reported for any other CDA. Concerning the pH and temperature optima, activity on chitin and chitosan polymers and activity on COS DP1-6, AngCDA is similar to already described CDAs 26,27,34,33 . We thus assume that our insight into this fungal CDA allows a deeper understanding of other CDAs of both bacterial and fungal origin.…”

“…So far, CDAs and other CE4 enzymes were mainly tested for their activity on different polymeric substrates like chitins, chitosans, acetyl xylans, peptidoglycans and their oligomeric counterparts. In recent years, for an increasing number of CE4 enzymes, the mode of action, referring to the different products that are generated over time, was investigated as well 2,4,25,26,27,28 . Computational methods, such as sequence and structure alignments as well as homology modelling and docking studies were used to complement these in vitro assays 2,27,28 .…”

“…In recent years, for an increasing number of CE4 enzymes, the mode of action, referring to the different products that are generated over time, was investigated as well 2,4,25,26,27,28 . Computational methods, such as sequence and structure alignments as well as homology modelling and docking studies were used to complement these in vitro assays 2,27,28 . To our knowledge, only a few CDAs were additionally studied by molecular dynamics (MD) simulations 22,29 and only for one CDA, from Cryptococcus laurentii, MD simulations were used for a more in depths analysis 30 .…”

Resistance Mechanisms to Antimicrobial Peptides in Gram-Positive Bacteria

“…In B. subtilis, deacetylation of the N-acetylmuramic acid by the protein PdaC confers resistance against lysozyme attack (Fukushima et al, 2005;Kobayashi et al, 2012;Grifoll-Romero et al, 2019). Another protein, PdgA described in L. monocytogenes is responsible for a similar resistance mechanism inducing N-acetylation of the peptidoglycan; a study by Popowska et al (2009) showed that a L. monocytogenes strain lacking PdgA was more susceptible to lysozyme and mutanolysin.…”

Preparation of Defined Chitosan Oligosaccharides Using Chitin Deacetylases

“…While it is theoretically possible to produce all pentamers in this way using seven different CDAs, some will be difficult to produce and their yields can be expected to be low, and most of them will require chromatographic purification. Also, for two of these enzymes, PcCDA and BsPdaC [ 38 , 39 ], N -acetylating activity in the presence of excess amounts of acetate has not yet been shown experimentally, but is assumed hypothetically here. For many of the pentamers, different production routes can be designed but for clarity, only the most feasible one is indicated in the figure.…”