Ionic network at the C‐terminus of the β‐glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: Functional role in the quaternary structure thermal stabilization

Abstract

Biochemical, crystallographic, and computational data support the hypothesis that electrostatic interactions are among the dominant forces in stabilizing hyperthermophilic proteins. The thermostable beta-glycosidase from the hyperthermophile Sulfolobus solfataricus (Ssbeta-gly) is an interesting model system for the study of protein adaptation to high temperatures. The largest ion-pair network of Ssbeta-gly is located at the tetrameric interface of the molecule; in this paper, key residues in this region were modified by site-directed mutagenesis and the stability of the mutants was analyzed by kinetics of thermal denaturation. All mutations produced faster enzyme inactivation, suggesting that the C-terminal ionic network prevents the dissociation into monomers, which is the limiting step in the mechanism of Ssbeta-gly inactivation. Moreover, the calculated reaction order showed that the mechanism of inactivation depends on the mutation introduced, suggesting that intermediates maintaining enzymatic activity are produced during the inactivation transition of some, but not all, mutants. Molecular models of each mutant allow us to rationalize the experimental evidence and give support to the current theories on the mechanism of ion pair stabilization in proteins from hyperthermophiles.

Discovery of hyperstable carbohydrate‐active enzymes through metagenomics of extreme environments

“…Interestingly, 14 enzymes found in Pool1 and 2 showed 100% identical amino acid sequence similarity to characterized glycosidases from S. solfataricus strains, namely, a β‐glycosidase (EC 3.2.1.21) (GH1), a β‐xylosidase/α‐L‐arabinosidase (EC 3.2.1.37/http://www.chem.qmul.ac.uk/iubmb/enzyme/3/2/1/55.html) (GH3), a cellulase (EC 3.2.1.4) and a xylanase (EC 3.2.1.151) (GH12), a maltooligosyltrehalose synthase (EC 5.4.99.15) and a glycogen debranching enzyme (EC 2.4.1.18) (GH13), a glucoamylase (EC 3.2.1.3) (GH15), an α‐L‐fucosidase (EC 3.2.1.51) (GH29), an α‐glucosidase (EC 3.2.1.20) and an α‐xylosidase (EC 3.2.1.77) (GH31), an α‐mannosidase (EC 3.2.1.24) (GH38), an α‐amylase (EC 3.2.1.1) (GH57), and a β‐glyco/xylosidase (EC 3.2.1.21/3.2.1.37) and a β‐ N ‐acetylglucosaminidase (EC 3.2.1.52) (GH116). These activities are involved in the hydrolysis and removal of sugar appendages of (hemi)cellulose polysaccharides, in starch/glycogen mobilization, and in the turnover of the oligosaccharides of N ‐glycosylated proteins . On the other hand, several GH families in Pool1 and 2 (GH2, GH65, GH78, GH99, GH130 in Table ) have no known characterized archaeal homologs.…”

“…52) (GH116). These activities are involved in the hydrolysis and removal of sugar appendages of (hemi)cellulose polysaccharides, in starch/glycogen mobilization, and in the turnover of the oligosaccharides of Nglycosylated proteins[49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65]. On the other hand, several GH families in Pool1 and 2 (GH2, GH65, GH78,…”

X‐ray structure of a membrane‐bound β‐glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii

“…(Although mesophilic BGBp has been reported to be octameric in solution,35 as well as in crystal,10 it may be considered as a tetramer of the dimers with similar interfaces to other dimeric β‐glycosidases 1211, 36, 37. However, for the monomeric BGPh, this is not the case.…”

Characterization of an acid-labile, thermostable β-glycosidase from Thermoplasma acidophilum

“…The amino acid sequence of S. solfataricus b-glycosidase exhibited 47% identity with enzyme herein, and the active site residues of both enzymes were highly conserved. The thermostability of b-glycosidase from S. solfataricus is determined by a network of ion pairs, located on the tetrameric surface, which results in destabilization of the enzyme under the basic conditions (D'Auria et al 1997(D'Auria et al , 1998Cobucci-Ponzano et al 2002). Similarly, the destabilization of the enzyme isolated in this study below pH 6.0 may be due to ion pair interactions on the surface of the dimer.…”