Directed Mutagenesis of Specific Active Site Residues on Fibrobacter succinogenes 1,3-1,4-β-D-Glucanase Significantly Affects Catalysis and Enzyme Structural Stability

Abstract

The functional and structural significance of amino acid residues Met 39, Glu56, Asp58, Glu60, and Gly63 of Fibrobacter succinogenes 1,3-1,4-β-D-glucanase was explored by the approach of site-directed mutagenesis, initial rate kinetics, fluorescence spectroscopy, and CD spectrometry. Glu56, Asp 58, Glu60, and Gly63 residues are conserved among known primary sequences of the bacterial and fungal enzymes. Kinetic analyses revealed that 240-, 540-, 570-, and 880-fold decreases in k cat were observed for the E56D, E60D, D58N, and D58E mutant enzymes, respectively, with a similar substrate affinity relative to the wild type enzyme. In contrast, no detectable enzymatic activity was observed for the E56A, E56Q, D58A, E60A, and E60Q mutants. These results indicated that the carboxyl side chain at positions 56 and 60 is mandatory for enzyme catalysis. M39F, unlike the other mutants, exhibited a 5-fold increase in Km value. Lower thermostability was found with the G63A mutant when compared with wild type or other mutant forms of F. succinogenes 1,3-1,4-β-D-glucanase. Denatured wild type and mutant enzymes were, however, recoverable as active enzymes when 8 M urea was employed as the denaturant. Structural modeling and kinetic studies suggest that Glu56, Asp58, and Glu 60 residues apparently play important role(s) in the catalysis of F. succinogenes 1,3-1,4-β-D-glucanase.