Roles of N287 in catalysis and product formation of amylomaltase from Corynebacterium glutamicum.
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.
['Nimpiboon|Pitchanan|P|', 'Krusong|Kuakarun|K|', 'Kaulpiboon|Jarunee|J|', 'Kidokoro|Shun-Ichi|S|', 'Pongsawasdi|Piamsook|P|']
D019943:Amino Acid Substitution D001216:Asparagine D001426:Bacterial Proteins D055162:Biocatalysis D020134:Catalytic Domain D003001:Cloning, Molecular D048230:Corynebacterium glutamicum D003500:Cyclization D003505:Cyclodextrins D004795:Enzyme Stability D004926:Escherichia coli D015870:Gene Expression D006004:Glycogen Debranching Enzyme System D006868:Hydrolysis D007700:Kinetics D007785:Lactose D016297:Mutagenesis, Site-Directed D009154:Mutation D009844:Oligosaccharides D017433:Protein Structure, Secondary D011994:Recombinant Proteins D013329:Structure-Activity Relationship D013379:Substrate Specificity
Amylomaltase Transglucosylation Cyclization Large-ring cyclodextrin Thermostability