Carbohydrate deacetylase, a key enzyme in oxidative chitin degradation, is evolutionarily linked to amino acid deacetylase.

The microbial oxidative cleavage of chitin, the second most abundant biopolymer in nature, generates a substantial amount of oxidized amino sugar, 2-(acetylamino)-2-deoxy-D-gluconic acid (GlcNAc1A). The catabolism of GlcNAc1A is key to the oxidative chitin degradation pathway. However, the molecular mechanism and evolution underlying this pathway remain elusive. Here, we target OngB, which initiates the GlcNAc1A catabolism, to explore the molecular mechanism driving the evolution of this process. We characterized PpOngB (the OngB from Pseudoalteromonas prydzensis ACAM 620) and its homologs as specific deacetylases for GlcNAc1A and solved the structures of WT PpOngB and its inactive mutant in complex with GlcNAc1A. Structural, mutational, and biochemical analyses revealed that PpOngB utilizes a D-aminoacylase-like (β/α)-barrel fold to deacetylate GlcNAc1A in a metal-dependent manner. PpOngB and its homologs significantly differ from other known carbohydrate de-N-acetylases in sequences, substrate specificities, and structures. Phylogenetic analysis indicated that PpOngB and its homologs represent a new carbohydrate de-N-acetylase family, forming a sister group of D-aminoacylases involved in the catabolism of N-acetyl-D-amino acids. Further structural analysis suggested that GlcNAc1A deacetylases likely evolved from an ancestral D-aminoacylase, undergoing structural and electrostatic modifications in the catalytic cavity to hydrolyze GlcNAc1A. This study provides insights into the catalytic mechanism and the divergent evolution of GlcNAc1A deacetylases, advancing our understanding of oxidative chitin degradation.