title: Structural basis of a mutant Y195I α-cyclodextrin glycosyltransferase with switched product specificity from α-cyclodextrin to β-/γ-cyclodextrin.
abstract: Cyclodextrin glycosyltransferase (EC 2.4.1.19) (CGTase) is an extracellular bacterial enzyme which has the unique capability of forming cyclodextrins from starch. Our previous investigation revealed that a mutant Y195I α-CGTase drastically altered the cyclodextrin specificity by switching toward the synthesis of both β- and γ-CDs (Xie et al., 2013a,b). In this study, we determined one X-ray structure of the mutant Y195I α-CGTase at 2.3Å. The overall structure was similar to that of the typical β-CGTase from Bacillus circulans 251, with minor difference in flexible domains since they showed about 70% homogeneity of amino acid sequences. The central site with isoleucine tended to be more flexible than tyrosine thus made the sugar chain, during the cyclization process, form a larger cyclodextrin like β- and γ-CDs surrounding the central site instead of α-CD. Superposition of the structure of Y195I α-CGTase with those of β-CGTase and γ-CGTase showed that residues Lys232, Lys89 and Arg177 at subsites +2, -3 and -7 could form smaller substrate binding cavity. In summary, the crystal structure revealed that moderate increase of mobility of the central site resulted in the switched product specificity from α-CD to β- and γ-CDs of the mutant Y195I α-CGTase. The space differences alongside the active domain may be another factor that impacts the product specificity of the CGTase.
authors.affiliation: State Key Laboratories of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Xie
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.
authors.forename: Yanjie
authors.initials: Y
authors.lastname: Hou
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.
authors.forename: Defeng
authors.initials: D
authors.lastname: Li
authors.affiliation: State Key Laboratories of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University (BUAA), Beijing 100191, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yue
authors.affiliation: State Key Laboratories of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Shijun
authors.initials: S
authors.lastname: Qian
authors.affiliation: State Key Laboratories of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: chaoyp@sun.im.ac.cn.
authors.forename: Yapeng
authors.initials: Y
authors.lastname: Chao
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003505:Cyclodextrins / Q000737:chemistry* / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D003505:Cyclodextrins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase
keywords: Crystal structure; Crystallization; Product specificity; Y195I α-CGTase