Structures of the Peptidoglycan N-Acetylglucosamine Deacetylase Bc1974 and Its Complexes with Zinc Metalloenzyme Inhibitors

Abstract

The cell wall peptidoglycan is recognized as a primary target of the innate immune system, and usually its disintegration results in bacterial lysis. Bacillus cereus, a close relative of the highly virulent Bacillus anthracis, contains 10 polysaccharide deacetylases. Among these, the peptidoglycan N-acetylglucosamine deacetylase Bc1974 is the highest homologue to the Bacillus anthracis Ba1977 that is required for full virulence and is involved in resistance to the host's lysozyme. These metalloenzymes belong to the carbohydrate esterase family 4 (CE4) and are attractive targets for the development of new anti-infective agents. Herein we report the first X-ray crystal structures of the NodB domain of Bc1974, the conserved catalytic core of CE4s, in the unliganded form and in complex with four known metalloenzyme inhibitors and two amino acid hydroxamates that target the active site metal. These structures revealed the presence of two conformational states of a catalytic loop known as motif-4 (MT4), which were not observed previously for peptidoglycan deacetylases, but were recently shown in the structure of a Vibrio clolerae chitin deacetylase. By employing molecular docking of a substrate model, we describe a catalytic mechanism that probably involves initial binding of the substrate in a receptive, more open state of MT4 and optimal catalytic activity in the closed state of MT4, consistent with the previous observations. The ligand-bound structures presented here, in addition to the five Bc1974 inhibitors identified, provide a valuable basis for the design of antibacterial agents that target the peptidoglycan deacetylase Ba1977.

The putative polysaccharide deacetylase Ba0331: cloning, expression, crystallization and structure determination

“…Acetate is one of the products of the deacetylation reaction, but its presence in the crystal structure of Ba0331 is most likely ascribable to impurities in the PEG crystallization medium (Blair et al, 2005). Similarly, in the previously reported homologous structures of Ba0330 (Arnaouteli et al, 2015) and Bc1974 (Giastas et al, 2018), which were determined at high resolution, acetate was also found bound to the zinc ion despite its absence from the initial constituents of the crystallization mixture. The near-octahedral coordination scheme of zinc is completed by a water molecule (Figs.…”

“…Specifically, MT1 and MT2 are involved in the immediate coordination of the active-site metal, MT3 and MT4 are located opposite to each other confining the binding site, while MT5 delimits the lower part of the binding groove (Arnaouteli et al, 2015;Giastas et al, 2018). In the catalytic groove (Figs.…”

“…A detailed comparative analysis of the groove-forming motifs between Ba0331 and its closest homologs, Ba0330 and Bc0361, has been described by Arnaouteli et al (2015), together with their influence on virulence. Additionally, a comparison of Ba0331, Ba0330, Bc0361 and Bc1974 at the sequence level has been presented by Giastas et al (2018).…”

“…6). Since no experimental data exist on the binding capabilities of the three putative polysaccharide deacetylases Ba0331, Ba0330 and Bc0361, a comparison of the structure of Ba0331 with the recently determined structure of the peptidoglycan GlcNAc deacetylase Bc1974, a metalloenzyme from B. cereus (Giastas et al, 2018) that is highly homologous to B. anthracis Ba1977 (97.1% sequence identity) and is required for the full virulence of the bacterium (Balomenou et al, 2013), reveals important information regarding the modified binding cavity. Like Ba0331, Bc1974 is also located in the extracellular space, being an N-terminally anchored membrane Electrostatic surface potential representation of Ba0331 (c) and comparison with those of Ba0330 (a), Bc0361 (b) and Bc1974 (d).…”

“…Most of these enzymes require a bivalent cation in their active site (Caufrier et al, 2003), which is generally coordinated by conserved aspartic acid and histidine residues (Blair et al, 2005). These bacterial metalloenzymes may represent a significant target for the ISSN 2053-230X # 2019 International Union of Crystallography structure-based design of molecules that block the immuneescape mechanism of bacteria (Blair et al, 2005;Giastas et al, 2018).…”