Site-selective CE4 enzyme from Rhodothermus marinus and diversified LmbE-Like catalysis in Bacillus cereus

Chitooligosaccharides (COS) display diverse biological functions that depend strongly on their N-acetylation pattern, motivating enzymatic approaches that enable regioselective deacetylation. Here, we identified a CE4 deacetylase, RmCBDA2510 from Rhodothermus marinus that performs strict, site-selective, and chain-length-dependent mono-deacetylation of COS. Exoglycosidase probing together with MALDI-TOF/TOF MS/MS showed that RmCBDA2510 precisely generates AD (GlcNAc-GlcN), ADA (GlcNAc-GlcN-GlcNAc), and AADA (GlcNAc-GlcNAc-GlcN-GlcNAc) from chitobiose, chitotriose, and chitotetraose, respectively, revealing a distinctive internal subsite preference. The enzyme is thermophilic and alkaline-active, and site-directed mutagenesis identified Asp41 as essential for catalysis, establishing RmCBDA2510 as a highly selective biocatalyst for producing structurally uniform, partially deacetylated COS. In contrast, the LmbE-like enzyme BCLmbE1534 from Bacillus cereus catalyzes both the deacetylation of N-acetylglucosamine glycosides and acylation/trans-acylation with short-chain fatty acids and biotin. This represents the first demonstration of trans-acylation within the LmbE family and expands its catalytic repertoire beyond simple deacetylation. Together, RmCBDA2510 and BCLmbE1534 provide complementary enzymatic platforms for tailoring COS structures and diversifying amino-sugar derivatives, thereby broadening the synthetic utility of carbohydrate-active deacetylases.