Peptidoglycan Deacetylases: Structural Basis for N-Acetylglucosamine and N-Acetylmuramic Acid Specificities and Biological Functions

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Peptidoglycan Deacetylases: Structural Basis for N-Acetylglucosamine and N-Acetylmuramic Acid Specificities and Biological Functions

Laia Grifoll-Romero 1 , María Angela Sainz-Polo 2 , David Albesa-Jové 2 , Marcelo Guerin 2 , Xevi Biarnés 1 , Antoni Planas 1

1 Institut Quimic de Sarria, University Ramon Llull, Barcelona, Spain, 2 CIC bioGUNE, Derio, Spain

The bacterial cell wall peptidoglycan (PG) is an elaborate polymeric mesh composed of a glycan chain of alternating β1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) units cross-linked via peptidyl bridges attached to the 3-O-lactoyl group of MurNAc residues. Pathogenic bacteria utilize acetylation (6-O-acetylation of MurNAc) and deacetylation (2-N-deacetylation of GlcNAc and/or MurNAc residues) of their cell wall PG to evade detection by the innate immune system. Deacetylation of MurNAc residues of PG is also involved in endospore formation and germination.

Peptidogycan deacetylases are members of family 4 carbohydrate esterases (CE4 enzymes), which also includes chitin deacetylases, poly-β1,6-N-acetylglucosamine deacetylases and some acetylxylan esterases. CE4 enzymes operate by a metal-assisted general acid/base catalytic mechanism [1]. A number of PG GlcNAc deacetylases have been characterized and their 3D structures determined by X-ray crystallography. They are specific for GlcNAc residues in the PG chain but they have also shown to deacetylate chitooligosaccharides, homo-oligomers of GlcNAc residues. In contrast, few PG MurNAc deaceylases have been biochemically and structurally characterized. The B. subtilis PdaA deacetylates MurNAc residues of peptidoglycan devoid of the peptide linked to the muramic acid 3-O-lactoyl group of MurNAc residues, and it is involved in sporulation to form muramic acid δ-lactam residues in the spore cortex peptidoglycan. It is specific for MurNAc residues and it is not active on chitooligosaccharides. Recently, a novel MurNAc deacetylase, BsPdaC, was identified [2], which deacetylates MurNAc residues of peptidoglycan, it is inactive on the glycan backbone devoid of peptidyl substitutions and, more strikingly, it also deacetylates chitooligosaccharides, an activity that was thought to be restricted to GlcNAc deacetylases.

To understand the molecular bases of such dual activity, we here report the biochemical characterization of BsPdaC, its mode of action on chitooligosaccharide substrates, the X-ray 3D structure of the CE4 catalytic domain, and structural comparison with canonical MurNAc and GlcNAc deacetylases [3]. We propose that PdaC is the first member of a new subclass of peptidoglycan MurNAc deacetylases, with yet unknown biological functions, based on these differential functional and structural characteristics. Additionally, and based on the novel specificity on chitooligosaccharides (COS), these PG deacetylases are potential biocatalysts in combination with chitin deacetylases for the synthesis of COS with defined patterns of acetylation, current targets for a number of biotech applications [4,5].

H.Aragunde, et al. (2018) Substrate recognition and specificity of chitin deacetylases and related family 4 carbohydrate esterases. Int. J. Mol. Sci. 19, 412

Kobayashi, K., et al.. (2012) Identification and characterization of a novel polysaccharide deacetylase C (PdaC) from Bacillus subtilis. J. Biol. Chem. 287, 9765–9776

L.Grifoll-Romero, et al. Structure-function relationships underlying the dual N-acetylmuramic and N-acetylglucosamine specificities of the peptidoglycan deacetylase PdaC from Bacillus subtilis. Submitted

Andrés, et al. Structural basis of chitin oligosaccharide deacetylation. Angew. Chem. Int. Ed. Engl. 53, 6882-6887 (2014)

L. Grifoll-Romero, et al., Chitin deacetylases: structures, specificities, and biotech applications. Polymers 10, 352 (2018).

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