Oligomerization as a strategy for cold adaptation: Structure and dynamics of the GH1 β-glucosidase from Exiguobacterium antarcticum B7

Abstract

Psychrophilic enzymes evolved from a plethora of structural scaffolds via multiple molecular pathways. Elucidating their adaptive strategies is instrumental to understand how life can thrive in cold ecosystems and to tailor enzymes for biotechnological applications at low temperatures. In this work, we used X-ray crystallography, in solution studies and molecular dynamics simulations to reveal the structural basis for cold adaptation of the GH1 β-glucosidase from Exiguobacterium antarcticum B7. We discovered that the selective pressure of low temperatures favored mutations that redesigned the protein surface, reduced the number of salt bridges, exposed more hydrophobic regions to the solvent and gave rise to a tetrameric arrangement not found in mesophilic and thermophilic homologues. As a result, some solvent-exposed regions became more flexible in the cold-adapted tetramer, likely contributing to enhance enzymatic activity at cold environments. The tetramer stabilizes the native conformation of the enzyme, leading to a 10-fold higher activity compared to the disassembled monomers. According to phylogenetic analysis, diverse adaptive strategies to cold environments emerged in the GH1 family, being tetramerization an alternative, not a rule. These findings reveal a novel strategy for enzyme cold adaptation and provide a framework for the semi-rational engineering of β-glucosidases aiming at cold industrial processes.

When substrate inhibits and inhibitor activates: implications of β-glucosidases

“…Such active site architecture has been proposed to limit the access of glucose to the glycone-binding site and confer the glucose tolerance [40]. Whatever is the underlying mechanism, the amino-acid residues responsible for the glucose tolerance are proposed to be located in the aglycone-binding site [40, 64, 65]. In the context of the effects of the nonproductive binding of substrate reported here, it is important that the structures of GH1 BGs show the presence of more than one aglycone-binding subsites [30, 40, 66–68].…”

The C-Domain of Oleuropein β-Glucosidase Assists in Protein Folding and Sequesters the Enzyme in Nucleus

“…It has been proposed that aggregated b-glucosidases exhibit enhanced enzymatic activity (Kim and Kim, 1998;Lee et al, 2006) and increased resistance against herbivore proteases (Gus-Mayer et al, 1994;Guirimand et al, 2010). Interestingly, oligomerization of a GH1 b-glucosidase from the psychrophilic prokaryote Exiguobacterium antarcticum B7 was correlated with cold adaptation (Zanphorlin et al, 2016). Taking into consideration that plants are obliged to recruit their defensive b-glucosidases in a wide temperature range, multimerization may also be a strategy to retain the activity at low temperatures.…”

Elucidating the regulation of glucose tolerance through the interaction between the reaction product and active site pocket residues of a β-glucosidase from Halothermothrix orenii

“…Similar mutations across H0HC94 and O08324 studied in our laboratory, seems to support the role of hydrophobic residues inside the active site pocket [12, 22]. Amino acid residues in the aglycone-binding site have been proposed to be responsible for glucose tolerance [13, 43]. Based on in-silico docking studies, Yang et al proposed that glucose tolerant β-glucosidase have a higher propensity of glucose binding near the middle of the pocket while less tolerant one can have glucose binding at the bottom [44].…”