title: Interaction of di-N-acetylchitobiosyl moranoline with a family GH19 chitinase from moss, Bryum coronatum.
abstract: Tri-N-acetylchitotriosyl moranoline, (GlcNAc)3-M, was previously shown to strongly inhibit lysozyme (Ogata M, Umemoto N, Ohnuma T, Numata T, Suzuki A, Usui T, Fukamizo T. 2013. A novel transition-state analogue for lysozyme, 4-O-β-tri-Nacetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate. J Biol Chem. 288:6072-6082). The findings prompted us to examine the interaction of di-N-acetylchitobiosyl moranoline, (GlcNAc)2-M, with a family GH19 chitinase from moss, Bryum coronatum (BcChi19A). Thermal unfolding experiments using BcChi19A and the catalytic acid-deficient mutant (BcChi19A-E61A) revealed that the transition temperature (Tm) was elevated by 4.3 and 5.8°C, respectively, upon the addition of (GlcNAc)2-M, while the chitin dimer, (GlcNAc)2, elevated Tm only by 1.0 and 1.4°C, respectively. By means of isothermal titration calorimetry, binding free energy changes for the interactions of (GlcNAc)3 and (GlcNAc)2-M with BcChi19A-E61A were determined to be -5.2 and -6.6 kcal/mol, respectively, while (GlcNAc)2 was found to interact with BcChi19A-E61A with markedly lower affinity. nuclear magnetic resonance titration experiments using (15)N-labeled BcChi19A and BcChi19A-E61A revealed that both (GlcNAc)2 and (GlcNAc)2-M interact with the region surrounding the catalytic center of the enzyme and that the interaction of (GlcNAc)2-M is markedly stronger than that of (GlcNAc)2 for both enzymes. However, (GlcNAc)2-M was found to moderately inhibit the hydrolytic reaction of chitin oligosaccharides catalyzed by BcChi19A (IC50 = 130-620 μM). A molecular dynamics simulation of BcChi19A in complex with (GlcNAc)2-M revealed that the complex is quite stable and the binding mode does not significantly change during the simulation. The moranoline moiety of (GlcNAc)2-M did not fit into the catalytic cleft (subsite -1) but was rather in contact with subsite +1. This situation may result in the moderate inhibition toward the BcChi19A-catalyzed hydrolysis.
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204, Nakamachi, Nara 631-8505, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Shinya
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204, Nakamachi, Nara 631-8505, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Urasaki
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204, Nakamachi, Nara 631-8505, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
authors.affiliation: Department of Bioscience and Biotechnology, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan.
authors.forename: Toki
authors.initials: T
authors.lastname: Taira
authors.affiliation: Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
authors.forename: Akari
authors.initials: A
authors.lastname: Suzuki
authors.affiliation: Department of Chemistry and Biochemistry, Fukushima National College of Technology, 30 Nagao, Iwaki, Fukushima 970-8034, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ogata
authors.affiliation: Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
authors.forename: Taichi
authors.initials: T
authors.lastname: Usui
authors.affiliation: Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu 90014, Finland.
authors.forename: Outi
authors.initials: O
authors.lastname: Lampela
authors.affiliation: Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu 90014, Finland.
authors.forename: André H
authors.initials: AH
authors.lastname: Juffer
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204, Nakamachi, Nara 631-8505, Japan fukamizo@nara.kindai.ac.jp.
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D017485:1-Deoxynojirimycin / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D020134:Catalytic Domain; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000378:metabolism*; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D009113:Muramidase / Q000037:antagonists & inhibitors / Q000737:chemistry; D011485:Protein Binding; D044003:Sphagnopsida / Q000737:chemistry
chemical_list: D004187:Disaccharides; D002686:Chitin; D017485:1-Deoxynojirimycin; C010913:N,N-diacetylchitobiose; D002688:Chitinases; D009113:Muramidase
keywords: NMR; chitin oligosaccharide; family GH19 chitinase; isothermal titration calorimetry; moranoline