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

Abstract

The beta-glycosidase of the hyperthermophilic Archaeon Pyrococcus horikoshii is a membrane-bound enzyme with the preferred substrate of alkyl-beta-glycosides. In this study, the unusual structural features that confer the extreme thermostability and substrate preferences of this enzyme were investigated by X-ray crystallography and docking simulation. The enzyme was crystallized in the presence of a neutral surfactant, and the crystal structure was solved by the molecular replacement method and refined at 2.5 A. The main-chain fold of the enzyme belongs to the (betaalpha)8 barrel structure common to the Family 1 glycosyl hydrolases. The active site is located at the center of the C-termini of the barrel beta-strands. The deep pocket of the active site accepts one sugar unit, and a hydrophobic channel extending radially from there binds the nonsugar moiety of the substrate. The docking simulation for oligosaccharides and alkylglucosides indicated that alkylglucosides with a long aliphatic chain are easily accommodated in the hydrophobic channel. This sparingly soluble enzyme has a cluster of hydrophobic residues on its surface, situated at the distal end of the active site channel and surrounded by a large patch of positively charged residues. We propose that this hydrophobic region can be inserted into the membrane while the surrounding positively charged residues make favorable contacts with phosphate groups on the inner surface of the membrane. The enzyme could thus adhere to the membrane in the proximity of its glycolipid substrate.

Purification and characterization of two extracellular endochitinases from Massilia timonae

“…The presence of a cluster of positively charged residues in the catalytic domain of the chitinase B from Serratia marcescens was reported by van Aalten et al 27 Similarly, some other glycosyl hydrolases contain positively charged patches that are thought to play a role in membrane adherence and substrate binding. 28,29 On the other hand, some bacterial chitinases seem to exhibit rather high levels of surface hydrophobicity. In a previous study, it was noticed that the chitinase from Bacillus sp.…”

Preparation of a glycosynthase from the β-glycosidase of the ArchaeonPyrococcus horikoshii

“…Among GH1 enzymes, Phob-gly has some peculiarities; it is monomeric, while the majority of the other members of the family is oligomeric, and the inspection of the 3D-structure suggested that it could be membrane-bound (Akiba et al 2004). In Scheme 1. addition, alkyl glucosides are good substrates of Phob-gly suggesting that the enzyme could be involved in the synthesis of glycolipids in vivo (Matsui et al 2000;Akiba et al 2004).…”

The Crystal Structure of Human Cytosolic β-Glucosidase Unravels the Substrate Aglycone Specificity of a Family 1 Glycoside Hydrolase

“…To date, the 3D structures of 18 GH1 β-glucosidases (in either resting, complexed, or transition state) have been determined from Eukaryota, [4][5][6][7][8][9][10][11][12] Bacteria [13][14][15][16][17][18] and Archaea. [19][20][21][22] Although the extent of sequence identity varies between 17% and 45% in the β-glucosidases, all the enzymes display a common (β/α) 8 -barrel motif. Apart from plant myrosinases and Klotho (KL) family members, all family 1 β-glucosidases conserve two catalytic glutamate residues located at the C-terminal end of β-strands 4 and 7.…”