title: Substrate recognition of N,N'-diacetylchitobiose deacetylase from Pyrococcus horikoshii.
abstract: 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.
authors.affiliation: National Institute of Advanced Industrial Science and Technology, Osaka 563-8577, Japan. Electronic address: nakamura-t@aist.go.jp.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Institute of Advanced Technology, Kindai University, Wakayama 649-6493, Japan.
authors.forename: Yasushige
authors.initials: Y
authors.lastname: Yonezawa
authors.affiliation: Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Tsuchiya
authors.affiliation: National Institute of Advanced Industrial Science and Technology, Osaka 563-8577, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Niiyama
authors.affiliation: National Institute of Advanced Industrial Science and Technology, Osaka 563-8577, Japan; Doshisha Women's College of Liberal Arts, Kyoto 602-0893, Japan.
authors.forename: Kurumi
authors.initials: K
authors.lastname: Ida
authors.affiliation: National Institute of Advanced Industrial Science and Technology, Osaka 563-8577, Japan.
authors.forename: Maki
authors.initials: M
authors.lastname: Oshima
authors.affiliation: Doshisha Women's College of Liberal Arts, Kyoto 602-0893, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Morita
authors.affiliation: National Institute of Advanced Industrial Science and Technology, Osaka 563-8577, Japan. Electronic address: k-uegaki@aist.go.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Uegaki
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D002686:Chitin / Q000031:analogs & derivatives; D048271:Chitosan; D018360:Crystallography, X-Ray; D004187:Disaccharides / Q000737:chemistry*; D006860:Hydrogen Bonding; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009844:Oligosaccharides; D010710:Phosphates / Q000737:chemistry; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D019843:Archaeal Proteins; D004187:Disaccharides; D009844:Oligosaccharides; D010710:Phosphates; C493484:oligochitosan; D002686:Chitin; C010913:N,N-diacetylchitobiose; D048271:Chitosan
keywords: 2-Deoxy-2-methylphosphoramido-d-glucose; Carbohydrate esterase family 14; Molecular dynamics; N,N′-diacetylchitobiose deacetylase; Pyrococcus horikoshii; X-ray crystallography