title: Molecular engineering of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040: structural determinants for the reaction product size and reactivity.
abstract: Cycloisomaltooligosaccharide glucanotransferase (CITase) is a member of glycoside hydrolase family 66 and it produces cycloisomaltooligosaccharides (CIs). Small CIs (CI-7-9) and large CIs (CI-≥10) are designated as oligosaccharide-type CIs (oligo-CIs) and megalosaccharide-type CIs (megalo-CIs) respectively. CITase from Bacillus circulans T-3040 (BcCITase) produces mainly CI-8 with little megalo-CIs. It has two family 35 carbohydrate-binding modules (BcCBM35-1 and BcCBM35-2). BcCBM35-1 is inserted in a catalytic domain of BcCITase and BcCBM35-2 is located at the C-terminal region. Our previous studies suggested that BcCBM35-1 has two substrate-binding sites (B-1 and B-2) [Suzuki et al. (2014) J. Biol. Chem. 289, 12040-12051]. We implemented site-directed mutagenesis of BcCITase to explore the preference for product size on the basis of the 3D structure of BcCITase. Mutational studies provided evidence that B-1 and B-2 contribute to recruiting substrate and maintaining product size respectively. A mutant (mutant-R) with four mutations (F268V, D469Y, A513V and Y515S) produced three times as much megalo-CIs (CI-10-12) and 1.5 times as much total CIs (CI-7-12) as compared with the wild-type (WT) BcCITase. The 3D structure of the substrate-enzyme complex of mutant-R suggested that the modified product size specificity was attributable to the construction of novel substrate-binding sites in the B-2 site of BcCBM35-1 and reactivity was improved by mutation on subsite -3 on the catalytic domain.
authors: *Applied Microbiology Division, National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba 305-8642, Japan.
authors: Ryuichiro
authors: R
authors: Suzuki
authors: ‡Biomolecular Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan.
authors: Nobuhiro
authors: N
authors: Suzuki
authors: ‡Biomolecular Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan.
authors: Zui
authors: Z
authors: Fujimoto
authors: ‡Biomolecular Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan.
authors: Mitsuru
authors: M
authors: Momma
authors: *Applied Microbiology Division, National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba 305-8642, Japan.
authors: Keitarou
authors: K
authors: Kimura
authors: ∥College of Life, Environment, and Advanced Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai 599-8531, Japan.
authors: Shinichi
authors: S
authors: Kitamura
authors: ¶Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nisi-9, Kita-ku, Sapporo 060-8589, Japan.
authors: Atsuo
authors: A
authors: Kimura
authors: *Applied Microbiology Division, National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba 305-8642, Japan.
authors: Kazumi
authors: K
authors: Funane
mesh_terms: D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D005964:Glucosyltransferases; C087517:cycloisomaltooligosaccharide glucanotransferase