title: Enzymatic properties of a GH19 chitinase isolated from rice lacking a major loop structure involved in chitin binding.
abstract: The catalytic domains of family GH19 chitinases have been found to consist of a conserved, α-helical core-region and different numbers (1-6) of loop structures, located at both ends of the substrate-binding groove and which extend over the glycon- and aglycon-binding sites. We expressed, purified and enzymatically characterized a GH19 chitinase from rice, Oryza sativa L. cv. Nipponbare (OsChia2a), lacking a major loop structure (loop III) connected to the functionally important β-stranded region. The new enzyme thus contained the five remaining loop structures (loops I, II, IV, V and C-term). The OsChia2a recombinant protein catalyzed hydrolysis of chitin oligosaccharides, (GlcNAc)n (n = 3-6), with inversion of anomeric configuration, indicating that OsChia2a correctly folded without loop III. From thermal unfolding experiments and calorimetric titrations using the inactive OsChia2a mutant (OsChia2a-E68Q), in which the catalytic residue Glu68 was mutated to glutamine, we found that the binding affinities towards (GlcNAc)n (n = 2-6) were almost proportional to the degree of polymerization of (GlcNAc)n, but were much lower than those obtained for a moss GH19 chitinase having only loop III [Ohnuma T, Sørlie M, Fukuda T, Kawamoto N, Taira T, Fukamizo T. 2011. Chitin oligosaccharide binding to a family GH19 chitinase from the moss, Bryum coronatum. FEBS J. 278:3991-4001]. Nevertheless, OsChia2a exhibited significant antifungal activity. It appears that loop III connected to the β-stranded region is important for (GlcNAc)n binding, but is not essential for antifungal activity.
authors.affiliation: Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara 631-8505, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Tanaka
authors.affiliation: Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara 631-8505, Japan.
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
authors.affiliation: Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara 631-8505, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D001665:Binding Sites; D020134:Catalytic Domain; D002686:Chitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D005658:Fungi / Q000472:pathogenicity; D006868:Hydrolysis; D009844:Oligosaccharides / Q000737:chemistry; D012275:Oryza / Q000737:chemistry / Q000201:enzymology* / Q000382:microbiology; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical / Q000235:genetics; D000072757:Protein Conformation, beta-Strand / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases
keywords: GH19 chitinase; chitin oligosaccharide; loop structure; pathogenesis-related protein; subsite structure