Mutational and structural studies of the active-site residues in truncated Fibrobacter succinogenes1,3-1,4-beta-D-glucanase.
1,3-1,4-beta-D-Glucanases (EC 3.2.1.73) specifically hydrolyze beta-1,4-glycosidic bonds located prior to beta-1,3-glycosidic linkages in lichenan or beta-D-glucans. It has been suggested that truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (TFsbeta-glucanase) can accommodate five glucose rings in its active site upon enzyme-substrate interaction. In this study, 12 mutant enzymes were created by mutating the conserved residues Gln70, Asn72, Gln81 and Glu85 proposed to bind to substrate subsites +1 and +2 and the catalytic properties of these mutants were determined. The most significant change in catalytic activity was observed on mutation of Gln70, with a 299-fold and 498-fold lower k(cat)/K(m) for the mutants Q70A and Q70I, respectively, compared with the wild-type enzyme. Mutagenesis, kinetic and structural studies revealed that the conserved residues surrounding the active site of TFsbeta-glucanase at substrate subsites +1 and +2 play an important role in its catalytic function, with the following order of importance: Gln70 > Asn72 > Glu85 > Gln81. The crystal structure of mutant E85I was determined at 2.2 A resolution. Further analysis of the E85I mutant structure revealed that the loop located at the concave site moved approximately 2 A from its position in the native enzyme complex without changing the core structure.
Tsai
Li-Chu
LC
Department of Molecular Science and Engineering, National Taipei University of Technology, Taipei 106, Taiwan. lichu@ntut.edu.tw
Huang
Hsiao-Chuan
HC
Hsiao
Ching-Hua
CH
Chiang
Yuan-Neng
YN
Shyur
Lie-Fen
LF
Lin
Yu-Shiun
YS
Lee
Shu-Hua
SH
D001665:Binding Sites; D020013:Calcium Signaling / Q000235:genetics; D002241:Carbohydrates / Q000737:chemistry; D020134:Catalytic Domain / Q000235:genetics*; D003001:Cloning, Molecular; D003460:Crystallization; D045848:Fibrobacter / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002241:Carbohydrates; D050505:Mutant Proteins; D006026:Glycoside Hydrolases; C028020:licheninase