caa a22 4500 606208186 CHVBK 20210128101021.0 cr unu---uuuuu 210128e20151001xx s 000 0 eng 10.1007/s00425-015-2330-4 doi (NATIONALLICENCE)springer-10.1007/s00425-015-2330-4 A class III chitinase without disulfide bonds from the fern, Pteris ryukyuensis [Elektronische Daten] crystal structure and ligand-binding studies [Yoshihito Kitaoku, Naoyuki Umemoto, Takayuki Ohnuma, Tomoyuki Numata, Toki Taira, Shohei Sakuda, Tamo Fukamizo] Main conclusion : We first solved the crystal structure of class III catalytic domain of a chitinase from fern (PrChiA-cat), and found a structural difference between PrChiA-cat and hevamine. PrChiA-cat was found to have reduced affinities to chitin oligosaccharides and allosamidin. Plant class III chitinases are subdivided into enzymes with three disulfide bonds and those without disulfide bonds. We here referred to the former enzymes as class IIIa chitinases and the latter as class IIIb chitinases. In this study, we solved the crystal structure of the class IIIb catalytic domain of a chitinase from the fern Pteris ryukyuensis (PrChiA-cat), and compared it with that of hevamine, a class IIIa chitinase from Hevea brasiliensis. PrChiA-cat was found to adopt an (α/β)8 fold typical of GH18 chitinases in a similar manner to that of hevamine. However, PrChiA-cat also had two large loops that extruded from the catalytic site, and the corresponding loops in hevamine were markedly smaller than those of PrChiA-cat. An HPLC analysis of the enzymatic products revealed that the mode of action of PrChiA-cat toward chitin oligosaccharides, (GlcNAc) n (n=4-6), differed from those of hevamine and the other class IIIa chitinases. The binding affinities of (GlcNAc)3 and (GlcNAc)4 toward the inactive mutant of PrChiA-cat were determined by isothermal titration calorimetry, and were markedly lower than those toward other members of the GH18 family. The affinity and the inhibitory activity of allosamidin toward PrChiA-cat were also lower than those toward the GH18 chitinases investigated to date. Several hydrogen bonds found in the crystal structure of hevamine-allosamidin complex were missing in the modeled structure of PrChiA-cat-allosamidin complex. The structural findings for PrChiA-cat successfully interpreted the functional data presented. Springer-Verlag Berlin Heidelberg, 2015 Allosamidin nationallicence Class III chitinase nationallicence Crystal structure nationallicence Isothermal titration calorimetry nationallicence Oligosaccharide hydrolysis nationallicence Pteris nationallicence PrChiA : A chitinase from Pteris ryukyuensis nationallicence PrChiA-cat : A class IIIb catalytic domain of PrChiA nationallicence (GlcNAc) n : β-1,4-Linked oligosaccharide of N-acetyl-d-glucosamine (GlcNAc) with a polymerization degree of n nationallicence ITC : Isothermal titration calorimetry nationallicence Kitaoku Yoshihito Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, 631-8505, Nara, Japan aut Umemoto Naoyuki Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, 631-8505, Nara, Japan aut Ohnuma Takayuki Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, 631-8505, Nara, Japan aut Numata Tomoyuki Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, 305-8566, Tsukuba, Ibaraki, Japan aut Taira Toki Department of Bioscience and Biotechnology, University of the Ryukyus, 1 Senbaru, 903-0213, Nishihara, Okinawa, Japan aut Sakuda Shohei Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, 113-8657, Tokyo, Japan aut Fukamizo Tamo Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, 631-8505, Nara, Japan aut Planta Springer Berlin Heidelberg 242/4(2015-10-01), 895-907 0032-0935 242:4<895 2015 242 425 https://doi.org/10.1007/s00425-015-2330-4 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00425-015-2330-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kitaoku Yoshihito Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, 631-8505, Nara, Japan aut NATIONALLICENCE 700 1- Umemoto Naoyuki Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, 631-8505, Nara, Japan aut NATIONALLICENCE 700 1- Ohnuma Takayuki Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, 631-8505, Nara, Japan aut NATIONALLICENCE 700 1- Numata Tomoyuki Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, 305-8566, Tsukuba, Ibaraki, Japan aut NATIONALLICENCE 700 1- Taira Toki Department of Bioscience and Biotechnology, University of the Ryukyus, 1 Senbaru, 903-0213, Nishihara, Okinawa, Japan aut NATIONALLICENCE 700 1- Sakuda Shohei Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, 113-8657, Tokyo, Japan aut NATIONALLICENCE 700 1- Fukamizo Tamo Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, 631-8505, Nara, Japan aut NATIONALLICENCE 773 0- Planta Springer Berlin Heidelberg 242/4(2015-10-01), 895-907 0032-0935 242:4<895 2015 242 425