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Structural and catalytic roles of amino acid residues located at substrate‐binding pocket in Fibrobacter succinogenes 1,3–1,4‐β‐ D ‐glucanase
Author(s) -
Chen JeHsin,
Tsai LiChu,
Huang HsiaoChuan,
Shyur LieFen
Publication year - 2010
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.22798
Subject(s) - fibrobacter succinogenes , glucanase , chemistry , enzyme , mutant , stereochemistry , catalysis , substrate (aquarium) , biochemistry , crystallography , biology , ecology , rumen , fermentation , gene
We created 12 mutant enzymes (E11L, F40I, Y42L, N44L, N44Q, E47I, L62G, K64A, K64M, R137M, R137Q, and N139A) from the truncated Fibrobacter succinogenes 1,3–1,4‐β‐ D ‐glucanase (TF‐glucanase). The enzymes were used to investigate the structural and catalytic roles of specific amino acid residues located at the catalytic pocket and having direct interactions with glucose subsites of the product β‐1,3–1,4‐cellotriose (CLTR). Fluorescence spectrometry showed no discernible changes in secondary structures among purified TF‐glucanase and the mutants. Kinetic analyses showed E11L, F40I, Y42L, R137M, and R137Q with a >10‐fold decrease of specific activity (11.2‐ to 67.4‐fold), and E11L, N44Q, E47I, K64M, R137M, R137Q, and N139A with a 2.17‐ to 4.3‐fold increase of K m value when compared with TF‐glucanase. Notably, E11L, R137Q, R137M, F40I, and N139A showed the most significant decrease in catalytic efficiency relative to TF‐glucanase, by 2155‐, 84.9‐, 48.5‐, 41.1‐, and 19.1‐fold, respectively; the five mutants showed the greatest changes in comparative energy ΔΔ G b , with values of 1.94 to 4.92 kcal/mol. Combined with results from kinetic and structure modeling analyses of all mutant enzymes and X‐ray crystallography of F40I, we elucidate that Glu11, Phe40, Arg137, and Asn139 play a crucial role in the catalysis of TF‐glucanase owing to their local and direct interaction through hydrogen bonds or van der Waals stacking interaction by aromatic rings onto the glucose subsites −3, −2, and −1 of CLTR/substrate. The overall globular structures in the wild‐type and mutant F40I enzymes do not differ. Proteins 2010. © 2010 Wiley‐Liss, Inc.