Chitin and peptidoglycan deacetylases: discovery, characterization and engineering


Grifoll Romero, Laia 


Chitin deacetylases (CDAs) and peptidoglycan deacetylases are members of family 4 carbohydrate esterases (CE4-enzymes), which also includes poly-β1,6-N-acetylglucosamine deacetylases and some acetylxylan esterases. Peptidoglycan is a major component of the bacterial cell wall. Pathogenic bacteria utilize acetylation (6-O-acetylation of MurNAc) and deacetylation (2-N-deacetylation of GlcNAc and/or MurNAc residues) of its own cell wall peptidoglycan to evade detection by the host’s innate immune system, with peptidoglycan deacetylases being proposed as novel antibacterial targets. Deacetylation of MurNAc residues of peptidoglycan is also involved in endospore formation and germination. Chitosans and chitooligosacharides (COS) are bioactive molecules with many current and potential new applications in several fields, and their physicochemical and biological properties are determined by their specific structures. There is a growing interest in developing selective enzymatic approaches to produce sequence-defined chitooligosaccharides to evaluate their biological functions and develop new applications, with CE4 enzymes showing great potential for their use as biocatalysts. Aiming to increase the available knowledge regarding the specificity of chitin and peptidoglycan deacetylases from CE4 family on different substrates and the sequential and structural features determining these specificities, this work reports the comprehensive characterization of the peptidoglycan deacetylase PdaC from Bacillus subtilis (BsPdaC), as well as the selection and characterization of novel CE4 enzymes. COS deacetylation kinetics has revealed that BsPdaC operates by a multiple-chain mechanism, with GlcNAc3 being the minimal substrate, and that the activity increases with the degree of glycan polymerization. The x-ray structure of the BsPdaC CE4 domain has been determined and the structural comparison with canonical MurNAc and GlcNAc deacetylases has led to propose that PdaC is the first member of a new subclass of peptidoglycan MurNAc deacetylases, with yet unknown biological functions, based on the differential functional and structural characteristics. Furthermore, the importance of key enzyme-substrate interactions has been highlighted by mutational analysis and the specificity and pattern of deacetylation has been altered by mutating a key substrate-interacting residue. The search for new CE4 enzymes based on a phylogenetic and bioinformatic analysis of the family is presented. Several new sequences have been selected and the recombinant proteins initially characterized, from which Tilletia controversa CDA has been identified as a novel CDA and Bacillus cereus Pda has been proposed as a novel MurNAc deacetylase for further characterization.




Antoni Planas Sauter 


IQS SE - Bioenginyeria

Date of defense