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

BackgroundFibrobacter succinogenes 1,3-1,4-β-d-glucanase (Fsβ-glucanase) is the only naturally occurring circularly permuted β-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 Fsβ-glucanase, corresponding to the major surface loop in the domain A region of Bacillus licheniformis glucanase.MethodsRational design approaches including site-directed mutagenesis, initial-rate kinetics, and structural modeling analysis were used in this study.ResultsOur 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 (kcat/KM) compared to the wild-type enzyme. The comparative energy ΔΔGb value in individual mutant enzymes was well correlated to their catalytic efficiency. D206R mutant enzyme exhibited the highest relative activity at 50 °C over 10 min, whereas K200F was the most heat-sensitive enzyme.ConclusionsThis 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 Fsβ-glucanase.General significanceResidues 200–209 in Fsβ-glucanase resided at the similar structural topology to that of Bacillus enzyme were found to play some similar catalytic function in glucanase.