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

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• 作者：Tsutomu Nakamura^a ; ^{nakamura-t@aist.go.jp" class="auth_mail" title="E-mail the corresponding author} ; Yasushige Yonezawa^b ; Yuko Tsuchiya^c ; Mayumi Niiyama^a ; ¹ ; Kurumi Ida^a ; ^d ; ² ; Maki Oshima^a ; Junji Morita^d ; Koichi Uegaki^a ; ^{k-uegaki@aist.go.jp" class="auth_mail" title="E-mail the corresponding author}
• 关键词：CE ; carbohydrate esterase ; Dac ; N ; N&prime ; -diacetylchitobiose deacetylase ; GlcN ; glucosamine ; GlcNAc ; N-acetylglucosamine ; GlcNAc2 ; N ; N&prime ; -diacetylchitobiose ; MD ; molecular dynamics ; MPG ; 2-deoxy-2-methylphosphoramido-d-glucose ; MshB ; N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-d-glucopyranoside deacetylase ; Pf-Dac ; N ; N&prime ; -diacetylchitobiose deacetylase from Pyrococcus furiosus ; Ph-Dac ; N ; N&prime ; -diacetylchitobiose deacetylase from Pyrococcus horikoshii ; VcCDA ; Vibrio cholerae chitin deac
• 刊名：Journal of Structural Biology
• 出版年：2016
• 出版时间：September 2016
• 年：2016
• 卷：195
• 期：3
• 页码：286-293
• 全文大小：2059 K

文摘

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.