Substrate recognition of N,N′-diacetylchitobiose deacetylase from Pyrococcus horikoshii

• Published in: Journal of structural biology 2016-09, Vol.195 (3), p.286-293
• Main Authors: Nakamura, Tsutomu, Yonezawa, Yasushige, Tsuchiya, Yuko, Niiyama, Mayumi, Ida, Kurumi, Oshima, Maki, Morita, Junji, Uegaki, Koichi
• Format: Article
• Language: English
• Subjects: 2-Deoxy-2-methylphosphoramido-d-glucose; Amino Acid Sequence; Archaeal Proteins - chemistry; Carbohydrate esterase family 14; Catalytic Domain; Chitin - analogs & derivatives; Crystallography, X-Ray; Disaccharides - chemistry; Hydrogen Bonding; Kinetics; Molecular dynamics; Molecular Dynamics Simulation; N,N′-diacetylchitobiose deacetylase; Phosphates - chemistry; Pyrococcus horikoshii; Pyrococcus horikoshii - enzymology; Substrate Specificity; X-ray crystallography
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1016/j.jsb.2016.07.015

Enzymes of carbohydrate esterase (CE) family 14 catalyze hydrolysis of N-acetyl groups at the non-reducing end of the N-acetylglucosamine (GlcNAc) residue of chitooligosaccharides or related compounds. N,N′-diacetylchitobiose deacetylase (Dac) belongs to the CE-14 family and plays a role in the chitinolytic pathway in archaea by deacetylating N,N′-diacetylchitobiose (GlcNAc2), which is the end product of chitinase. In this study, we revealed the structural basis of reaction specificity in CE-14 deacetylases by solving a crystal structure of Dac from Pyrococcus horikoshii (Ph-Dac) in complex with a novel reaction intermediate analog. We developed 2-deoxy-2-methylphosphoramido-d-glucose (MPG) as the analog of the tetrahedral oxyanion intermediate of the monosaccharide substrate GlcNAc. The crystal structure of Ph-Dac in complex with MPG demonstrated that Arg92, Asp115, and His152 side chains interact with hydroxyl groups of the glucose moiety of the non-reducing-end GlcNAc residue. The amino acid residues responsible for recognition of the MPG glucose moiety are spatially conserved in other CE-14 deacetylases. Molecular dynamics simulation of the structure of the Ph-Dac-GlcNAc2 complex indicated that the reducing GlcNAc residue is placed in a large intermolecular cleft and is not involved with specific interactions with the enzyme. This observation was consistent with results indicating that Ph-Dac displayed similar kinetic parameters for both GlcNAc and GlcNAc2. This study provides the structural basis of reaction-site specificity of Dac and related CE-14 enzymes.