title: Roles of N287 in catalysis and product formation of amylomaltase from Corynebacterium glutamicum.
abstract: Amylomaltase catalyzes intermolecular and intramolecular transglucosylation reactions to form linear and cyclic oligosaccharides, respectively. The aim of this work is to investigate the structure-function relationship of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM). Site-directed mutagenesis was performed to substitute Tyr for Asn287 (N287Y) to determine its role in controlling amylomaltase activity and product formation. Expression of the wild-type (WT) and N287Y was achieved by cultivating recombinant cells in the medium containing lactose at 16 °C for 14 h. The purified mutated enzyme showed a significant decrease in all transglucosylation activities while hydrolysis activity was not changed. Optimum temperature and pH for disproportionation reaction were slightly changed upon mutation while those for cyclization reaction were not changed. Interestingly, N287Y showed a change in large-ring cyclodextrin (LR-CD) product profile in which the larger size was observed together with an increase in thermostability and substrate preference for G5 in addition to G3. The secondary structure of the mutated enzyme was slightly changed in related to the WT as evidenced from circular dichroism analysis. This work thus demonstrates that N287 is required for transglucosylation activities of CgAM. Having an aromatic residue in this position increased thermostability, changed product profile and substrate preference but demolished most enzyme activities.
authors: Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand; Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok, 10330, Thailand.
authors: Pitchanan
authors: P
authors: Nimpiboon
authors: Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok, 10330, Thailand.
authors: Kuakarun
authors: K
authors: Krusong
authors: Department of Pre-clinical Science (Biochemistry), Faculty of Medicine, Thammasat University, Pathumthani, 12120, Thailand.
authors: Jarunee
authors: J
authors: Kaulpiboon
authors: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, 940-2188, Japan.
authors: Shun-Ichi
authors: S
authors: Kidokoro
authors: Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok, 10330, Thailand. Electronic address: piamsook.p@chula.ac.th.
authors: Piamsook
authors: P
authors: Pongsawasdi
mesh_terms: D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D048230:Corynebacterium glutamicum / Q000737:chemistry* / Q000201:enzymology; D003500:Cyclization; D003505:Cyclodextrins / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D003505:Cyclodextrins; D006004:Glycogen Debranching Enzyme System; D009844:Oligosaccharides; D011994:Recombinant Proteins; D001216:Asparagine; C020569:4 alpha-glucanotransferase; D007785:Lactose
keywords: Amylomaltase; Cyclization; Large-ring cyclodextrin; Thermostability; Transglucosylation