Structural and catalytic roles of residues located in beta13 strand and the following beta-turn loop in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase.
BACKGROUND
Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fsbeta-glucanase) is the only naturally occurring circularly permuted beta-glucanase among bacterial glucanases with reverse protein domains. We characterized the functional and structural significance of residues 200-209 located in the domain B of Fsbeta-glucanase, corresponding to the major surface loop in the domain A region of Bacillus licheniformis glucanase.


METHODS
Rational design approaches including site-directed mutagenesis, initial-rate kinetics, and structural modeling analysis were used in this study.


RESULTS
Our kinetic data showed that D202N and D206N exhibited a 1.8- and 1.5-fold increase but G207N, G207-, F205L, N208G and T204F showed a 7.0- to 2.2-fold decrease, in catalytic efficiency (k(cat)/K(M)) compared to the wild-type enzyme. The comparative energy DeltaDeltaG(b) value in individual mutant enzymes was well correlated to their catalytic efficiency. D206R mutant enzyme exhibited the highest relative activity at 50 degrees C over 10 min, whereas K200F was the most heat-sensitive enzyme.


CONCLUSIONS
This study demonstrates that Phe205, Gly207, and Asn208 in the Type II turn of the connecting loop may play a role in the catalytic function of Fsbeta-glucanase.


CONCLUSIONS
Residues 200-209 in Fsbeta-glucanase resided at the similar structural topology to that of Bacillus enzyme were found to play some similar catalytic function in glucanase.
Lin
Yu-Shiun
YS
Agricultural Biotechnology Research Center, Academia Sinica, Nankang, Taipei 115, Taiwan, ROC.
Tsai
Li-Chu
LC
Lee
Shu-Hua
SH
Yuan
Hanna S
HS
Shyur
Lie-Fen
LF
D000595:Amino Acid Sequence; D002384:Catalysis; D045848:Fibrobacter / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D006026:Glycoside Hydrolases; C028020:licheninase