Site-saturation mutagenesis of central tyrosine 195 leading to diverse product specificities of an α-cyclodextrin glycosyltransferase from Paenibacillus sp. 602-1.
Central tyrosine 195 plays an important role in the active site of cyclodextrin glycosyltransferase (CGTase) that is highly conservative among various CGTases. However, a detailed functional understanding of this subsite is lacking. In this study, we applied site-directed saturation mutagenesis to investigate the effect of tyrosine 195 on the hydrolytic activity and cyclization specificity of an α-CGTase. A total of 17 mutant CGTases were obtained and heterologously expressed in E. coli. The mutant Y195F α-CGTase showed similar characteristics with wild-type α-CGTase. The other mutant α-CGTases showed considerably lower activity for starch-degradation and cyclodextrin (CD) formation. Interestingly, we found that the main product of mutant Y195R α-CGTase was γ-CDs (50%), not α-CDs (35%). The mutant Y195I α-CGTase drastically altered the CD specificity of α-CGTase, which showed a switch toward the synthesis of both β- and γ-CDs with percentages of 34% and 38%, respectively. Other mutant CGTases retained the α-CD as the main product but with lower percentages than wild-type α-CGTase. Mutant Y195F, Y195I, and Y195R CGTases showed an optimal temperature of 50°C and pH 6.5. The mutants Y195I and Y195R also showed better thermostability. These findings suggested that aromatic amino acids Tyr or Phe at the 195 position were important for the amylolytic activity and cyclization specificity of α-CGTase. The mutants Y195I CGTase and Y195R CGTase have potential applications for γ-CD production in the future.
State Key Laboratories of Transducer Technology, National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Ting
T
Xie
State Key Laboratories of Transducer Technology, National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Binghong
B
Song
State Key Laboratories of Transducer Technology, National Engineering Lab for Industrial Enzymes, 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.
Yang
Y
Yue
State Key Laboratories of Transducer Technology, National Engineering Lab for Industrial Enzymes, 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.
Yapeng
Y
Chao
State Key Laboratories of Transducer Technology, National Engineering Lab for Industrial Enzymes, 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.
Shijun
S
Qian
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003500:Cyclization; D003505:Cyclodextrins / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D010649:Phenylalanine / Q000235:genetics*; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014443:Tyrosine / Q000235:genetics*
D001426:Bacterial Proteins; D003505:Cyclodextrins; D014443:Tyrosine; D010649:Phenylalanine; D013213:Starch; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase
Central site Tyr195; Cyclodextrin; Cyclodextrin glycosyltransferases; Product specificity; Site-directed saturation mutagenesis