Received: September 4, 2021. Revised: November 1, 2021. Accepted: November 1, 2021 © The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com Glycobiology, 2022, 32, 4, 356–364 https://doi.org/10.1093/glycob/cwab117 Advance access publication date 22 November 2021 Original Article Editor’s Choice A conserved loop structure of GH19 chitinases assists the enzyme function from behind the core-functional region Daiki Kawamoto 2 , Tomoya Takashima 2, † , Tamo Fukamizo 2,1 , Tomoyuki Numata 3,4 , Takayuki Ohnuma 2,5,* 2 Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara 631-8505, Japan, 3 Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan, 4 Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 Japan, 5 Agricultural Technology and Innovation Research Institute (ATIRI), Kindai University, 3327-204 Nakamachi, Nara 631-8505, Japan *To whom correspondence should be addressed: Tel: +81-742-43-7297; Fax: +81-742-43-8976; e-mail: ohnumat@nara.kindai.ac.jp (Takayuki Ohnuma); Tel: +81-742-43-7297; Fax: +81-742-43-8976; e-mail: tamo0111fuka@gmail.com (Tamo Fukamizo) † Present address: Laboratory of Pharmaceutics, Faculty of Pharmacy, Osaka Ohtani University, Osaka, Japan Plant GH19 chitinases have several loop structures, which may define their enzymatic properties. Among these loops, the longest loop, Loop-III, is most frequently conserved in GH19 enzymes. A GH19 chitinase from the moss Bryum coronatum (BcChi-A) has only one loop structure, Loop-III, which is connected to the catalytically important β-sheet region. Here, we produced and characterized a Loop-III-deleted mutant of BcChi-A (BcChi-A-III) and found that its stability and chitinase activity were strongly reduced. The deletion of Loop-III also moderately affected the chitooligosaccharide binding ability as well as the binding mode to the substrate-binding groove. The crystal structure of an inactive mutant of BcChi-A-III was successfully solved, revealing that the remaining polypeptide chain has an almost identical fold to that of the original protein. Loop-III is not necessarily essential for the folding of the enzyme protein. However, closer examination of the crystal structure revealed that the deletion of Loop-III altered the arrangement of the catalytic triad, Glu61, Glu70 and Ser102, and the orientation of the Trp103 side chain, which is important for sugar residue binding. We concluded that Loop-III is not directly involved in the enzymatic activity but assists the enzyme function by stabilizing the conformation of the β-sheet region and the adjacent substrate-binding platform from behind the core-functional regions. Key words: chitooligosaccharide binding; crystal structure; GH19 chitinase; isothermal titration calorimetry; loop structure. Introduction Chitin is a β -1,4-linked polysaccharide of N-acetylglucosamine (GlcNAc) and is present in crustacean shells, insect cuticles and fungal cell walls. Chitinases hydrolyze the β -1,4- glycosidic linkages in chitin, producing chitooligosaccharides. Based on their amino acid sequences, chitinases are classified into two families, GH18 and GH19 (http://www.cazy.org/; Henrissat and Davies 1997). GH18 chitinases are widely distributed in living organisms, whereas GH19 enzymes are found only in plants and some bacteria (Donnelly and Barnes 2004; Kawase et al. 2004; Duo-Chuan 2006; Bhattacharya et al. 2007; Arakane and Muthukrishnan 2010). Plant chitinases have various physiological functions, including self- defense, growth and stress tolerance (Kasprzewska 2003). The multiplicity in their functions may be related to the variation in their structures. In fact, the members of the GH19 plant chitinases are further subdivided into several classes, according to their domain organization and loop arrangements, which may define their enzymatic properties as well as their physiological functions (Arakane et al. 2012). It is highly desirable to study the role of these loops in their functions to understand the relationship between the structure and physiological functions. The crystal structure of a GH19 chitinase from barley seeds was reported by Hart et al. (1995) (Supplementary Figure 1). This was the first three-dimensional structure of an enzyme of this GH family. Barley chitinase has a core structure com- posed of two α-helical domains. In addition, four smaller loop structures are located at both ends of the substrate- binding groove lying in between the two domains, and a major loop structure, Loop-III, is located behind the glycon-binding groove (negatively numbered subsites). Figure 1 shows a mul- tiple sequence alignment of four GH19 chitinases from plants, indicating the locations of the individual loops, designated as Loop-I, -II, -III, -IV, -V and -VI from the N-terminus. A GH19 chitinase from rye seeds (RSC-c), whose sequence is aligned at the top of the sequence alignment, was almost identical to the barley chitinase with respect to their struc- tures (Ohnuma et al. 2002). GH19 chitinases lacking four loops (Loop-I, -II, -V and -VI or Loop-II, -IV, V and VI) at both ends of the binding groove (“loopless” chitinases) were isolated from two Streptomyces species and the evergreen conifer Norway spruce, respectively (Hoell et al. 2006; Kezuka et al. 2006; Ubhayasekera et al. 2009). It was suggested that the substrate-binding grooves of the loopless enzymes are shorter than those of “loopful” enzymes, such as GH19 Downloaded from https://academic.oup.com/glycob/article/32/4/356/6433007 by guest on 01 May 2023