Cell signaling and other biological activities of chitooligosaccharides (COSs) seem to be dependent not only on the degree of polymerization, but markedly on the specific de-N-acetylation pattern. Chitin de-N-acetylases (CDAs) catalyze the hydrolysis of the acetamido group in GlcNAc residues of chitin, chitosan, and COS. A major challenge is to understand how CDAs specifically define the distribution of GlcNAc and GlcNH2 moieties in the oligomeric chain. We report the crystal structure of the Vibrio cholerae CDA in four relevant states of its catalytic cycle. The two enzyme complexes with chitobiose and chitotriose represent the first 3D structures of a CDA with its natural substrates in a productive mode for catalysis, thereby unraveling an induced-fit mechanism with a significant conformational change of a loop closing the active site. We propose that the deacetylation pattern exhibited by different CDAs is governed by critical loops that shape and differentially block accessible subsites in the binding cleft of CE4 enzymes.

Background: Glycoglycerolipids, synthesized by glycosyltransferases (glycolipid synthases), are key structural membrane components in mycoplasma. Results: M. genitalium GT MG517 has been recombinantly expressed and kinetically characterized. It is a processive GT transferring glucosyl or galactosyl residues to membrane-bound diacylglycerol, with higher monoglycosyl-than diglycosyltransferase activity. Inhibition of the enzyme results in mycoplasma growth inhibition. Conclusion: The membrane glycoglycerolipids of M. genitalium are synthesized by a single processive glycosyltransferase, GT MG517. It is proposed as a potential therapeutic target against mycoplasma infections. Significance: The essential function of GT MG517 for mycoplasma viability and the absence of glycoglycerolipids in animal host cells of mycoplasma infections make the enzyme a target for drug discovery by designing specific inhibitors.

Cell signaling and other biological activities of chitooligosaccharides (COSs) seem to be dependent not only on the degree of polymerization, but markedly on the specific de‐N‐acetylation pattern. Chitin de‐N‐acetylases (CDAs) catalyze the hydrolysis of the acetamido group in GlcNAc residues of chitin, chitosan, and COS. A major challenge is to understand how CDAs specifically define the distribution of GlcNAc and GlcNH2 moieties in the oligomeric chain. We report the crystal structure of the Vibrio cholerae CDA in four relevant states of its catalytic cycle. The two enzyme complexes with chitobiose and chitotriose represent the first 3D structures of a CDA with its natural substrates in a productive mode for catalysis, thereby unraveling an induced‐fit mechanism with a significant conformational change of a loop closing the active site. We propose that the deacetylation pattern exhibited by different CDAs is governed by critical loops that shape and differentially block accessible subsites in the binding cleft of CE4 enzymes.