Directed Mutagenesis of Specific Active Site Residues onFibrobacter succinogenes1,3–1,4-β-d-Glucanase Significantly Affects Catalysis and Enzyme Structural Stability

Jui-Lin Chen,Li-Chu Tsai,Tuan-Nan Wen,Jyh-Bing Tang,Hanna S Yuan,Lie-Fen Shyur

doi:10.1074/jbc.m100843200

Abstract

The functional and structural significance of amino acid residues Met(39), Glu(56), Asp(58), Glu(60), and Gly(63) of Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase was explored by the approach of site-directed mutagenesis, initial rate kinetics, fluorescence spectroscopy, and CD spectrometry. Glu(56), Asp(58), Glu(60), and Gly(63) 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 K(m) 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-beta-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 Glu(56), Asp(58), and Glu(60) residues apparently play important role(s) in the catalysis of F. succinogenes 1,3-1,4-beta-d-glucanase.

Highlights

1,3–1,4-␤-D-Glucanase (1,3–1,4-␤-D-glucan 4-glucanohydrolases, EC 3.2.1.73; lichenase) hydrolyzes 1,4-␤-Dglycosidic bonds adjacent to 1,3-␤-linkages in mixed linked ␤-glucans, yielding mainly cellobiosyltriose and cellotriosyltetraose [1]
The functional and structural significance of amino acid residues Met39, Glu56, Asp58, Glu60, and Gly63 of Fibrobacter succinogenes 1,3–1,4-␤-D-glucanase was explored by the approach of site-directed mutagenesis, initial rate kinetics, fluorescence spectroscopy, and CD spectrometry
Bacterial 1,3–1,4-␤-D-glucanases belong to the category of retaining enzymes and are classified into family 16 endoglucanases [3]

Summary

EXPERIMENTAL PROCEDURES

Subcloning of F. succinogenes 1,3–1,4-␤-D-Glucanase Gene—The fulllength cDNA of F. succinogenes 1,3–1,4-␤-D-glucanase (Fs␤-glucanase) in a pIJ10 plasmid was amplified and introduced with NcoI and EcoRI restriction enzyme recognition sites at the 5Ј and 3Ј ends, respectively, by using a PCR-based method. The recombinant gene sequence for Fs␤-glucanase, designated “pFsNcE,” was confirmed by DNA sequencing using the chain termination method [17]. In this DNA construct, a pelB leading peptide at the N terminus plus 19 extra amino acid residues including a His tag at the C terminus to facilitate protein purification were included. Site-directed Mutagenesis—Plasmid pFsNcE was used as the template for a PCR-based mutation [18] of Fs␤-glucanase. For this purpose, a pair of complementary mutagenic primers was designed for the mutation of each desired amino acid residue. The mutagenic PCR reaction mixture consisted of 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl, pH 8.8, 2 mM MgSO4, 0.1% Triton X-100, 0.1 mg/ml nuclease-free bovine serum albumin, 10 ng of template DNA, 0.5 mM dNTPs, 0.5 ␮M each of the complementary primers, and 2.5 units of cloned Turbo Pfu DNA polymerase

The abbreviations used are

RESULTS

DISCUSSION