Acetylation Activity and Specificity of Peptidoglycan Deacetylase Bs PdaC Acting in Reverse on Chitooligosaccharides

Partially acetylated chitosan oligosaccharides play key biological roles and have numerous applications, with their bioactivities being strongly influenced by the acetylation pattern of their glucosamine monomers. However, chemical de‐N‐acetylation reactions of chitooligosaccharides (COS) lack the regioselectivity required to control these patterns. Chitin deacetylases (CDAs) exhibit diverse deacetylation patterns, becoming powerful biocatalysts for the tailored production of functional partially‐acetylated chitosan oligosaccharides. However, known CDAs do not cover all necessary patterns. CDAs have also been shown to catalyze the reverse reaction by acetylating glucosamine oligomers in the presence of excess acetate, which complements the portfolio of sequence‐defined partially acetylated chitosan oligomers (paCOS). In this study, the regioselectivity of the peptidoglycan de‐ N ‐acetylase Bs PdaC from Bacillus subtilis during the N ‐acetylation of chitopentaose (GlcNH 2 ) 5 (D5) was investigated. Remarkably, the enzyme is able to acetylate all but the reducing end GlcNH 2 units, mirroring the natural deacetylation reaction, giving access to new patterns of acetylation not found with known CDAs. The enzyme primarily produced A3D2 and A4D1 (A: GlcNAc, D: GlcNH 2 ), and MS sequencing revealed a novel acetylation pattern (DAAAD and AAAAD). Different conditions were tested, but the product profile remained unchanged. This new specificity, producing AAAAD, expands the diversity of sequence‐defined paCOS to evaluate their bioactivities.