Structural and catalytic roles of residues located in β13 strand and the following β-turn loop in Fibrobacter succinogenes 1,3-1,4-β-d-glucanase
Resource
Biochimica et Biophysica Acta (BBA) - General Subjects, 1790(4), 231-239
Journal
Biochimica et Biophysica Acta (BBA)-General Subjects
Pages
231-239
Date Issued
2009
Date
2009
Author(s)
Lin, Yu-Shiun
Tsai, Li-Chu
Lee, Shu-Hua
Yuan, Hanna S.
Shyur, Lie-Fen
Abstract
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. Rational design approaches including site-directed mutagenesis, initial-rate kinetics, and structural modeling analysis were used in this study. 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. 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. 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.
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