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

Leticia Maria Zanphorlin,Juliana Fattori,Elaine Crespim,Celisa Caldana Costa Tonoli,Priscila Oliveira De Giuseppe,Mario Tyago Murakami,Roberto Ruller,Rodrigo Vargas Honorato,Paulo Sergio Lopes De Oliveira

doi:10.1038/srep23776

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.

Highlights

Β -glucosidases are key enzymes in cellulose depolymerization, acting synergistically with endoglucanases and cellobiohydrolases. They catalyze the breakdown of cellobiose and short cello-oligomers into glucose, the final step of cellulose saccharification. β -glucosidases with high activity at low and moderate temperatures have been of much interest for flavor enhancement in wines[6,7] and production of cellulosic biofuels via simultaneous saccharification and fermentation[8] (SSF)–a process in which cell wall-degrading enzymes operate at typical fermenting conditions of Saccharomyces cerevisiae
The elucidation of the molecular adaptations that rendered this enzyme cold active and glucose tolerant expands our current understanding about enzyme cold adaptation and provides structural data to support the semi-rational design of high-performance β -glucosidases for biotechnological applications
We found that the molecular strategies that lead EaBglA to be cold-active targeted superficial residues from peripheral helices and loops, with a trend to replace charged by neutral amino acids

Summary

Introduction

Β -glucosidases are key enzymes in cellulose depolymerization, acting synergistically with endoglucanases and cellobiohydrolases. Β -glucosidases with high activity at low and moderate temperatures have been of much interest for flavor enhancement in wines[6,7] and production of cellulosic biofuels via simultaneous saccharification and fermentation[8] (SSF)–a process in which cell wall-degrading enzymes operate at typical fermenting conditions of Saccharomyces cerevisiae. In this study, we aimed to shed light on the structural basis for cold adaptation and glucose tolerance of EaBglA For this purpose, we combined X-ray crystallography, in solution studies and molecular dynamics simulations to find out structural features that allowed this enzyme to adapt to the low temperatures of Antarctic lakes. The elucidation of the molecular adaptations that rendered this enzyme cold active and glucose tolerant expands our current understanding about enzyme cold adaptation and provides structural data to support the semi-rational design of high-performance β -glucosidases for biotechnological applications

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