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

Zanphorlin LM, de Giuseppe PO, Honorato RV, Tonoli CC, Fattori J, Crespim E, de Oliveira PS, Ruller R, Murakami MT - Sci Rep (2016)

Bottom Line: Psychrophilic enzymes evolved from a plethora of structural scaffolds via multiple molecular pathways.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.The tetramer stabilizes the native conformation of the enzyme, leading to a 10-fold higher activity compared to the disassembled monomers.

View Article: PubMed Central - PubMed

Affiliation: Brazilian Bioethanol Science and Technology Laboratory, Campinas, São Paulo, Brazil.

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.

No MeSH data available.


Related in: MedlinePlus

The unique tetrameric structure of EaBglA.(A) The crystal structures of EaBglA reveal a tetrameric arrangement with 222 symmetry. The two independent tetramers observed in the P21 and C2221 crystals (multicolor and gray) are in slightly different conformations, suggesting inter-subunits motions (arrows). (B) Two pairs of interfacing active sites (1–2 and 3–4) locate at opposite faces of the tetramer. SAXS profile (C) and ab-initio model (D) agree with the theoretical curve and high-resolution structure of the tetramer. (E) AUC assays show the presence of tetramers in solution, even at low protein concentrations.
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f2: The unique tetrameric structure of EaBglA.(A) The crystal structures of EaBglA reveal a tetrameric arrangement with 222 symmetry. The two independent tetramers observed in the P21 and C2221 crystals (multicolor and gray) are in slightly different conformations, suggesting inter-subunits motions (arrows). (B) Two pairs of interfacing active sites (1–2 and 3–4) locate at opposite faces of the tetramer. SAXS profile (C) and ab-initio model (D) agree with the theoretical curve and high-resolution structure of the tetramer. (E) AUC assays show the presence of tetramers in solution, even at low protein concentrations.

Mentions: The two crystallographic structures of EaBglA, solved in distinct space groups, presented the same tetrameric arrangement (Fig. 2A). Structural comparisons revealed that the EaBglA subunits assemble in a different way from those of GH1 tetramers available at the Protein Data Bank (PDB), indicating that the tetrameric arrangement of EaBglA is the first of its kind to be reported within the GH1 family (Supplementary Fig. S2).


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

Zanphorlin LM, de Giuseppe PO, Honorato RV, Tonoli CC, Fattori J, Crespim E, de Oliveira PS, Ruller R, Murakami MT - Sci Rep (2016)

The unique tetrameric structure of EaBglA.(A) The crystal structures of EaBglA reveal a tetrameric arrangement with 222 symmetry. The two independent tetramers observed in the P21 and C2221 crystals (multicolor and gray) are in slightly different conformations, suggesting inter-subunits motions (arrows). (B) Two pairs of interfacing active sites (1–2 and 3–4) locate at opposite faces of the tetramer. SAXS profile (C) and ab-initio model (D) agree with the theoretical curve and high-resolution structure of the tetramer. (E) AUC assays show the presence of tetramers in solution, even at low protein concentrations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4815018&req=5

f2: The unique tetrameric structure of EaBglA.(A) The crystal structures of EaBglA reveal a tetrameric arrangement with 222 symmetry. The two independent tetramers observed in the P21 and C2221 crystals (multicolor and gray) are in slightly different conformations, suggesting inter-subunits motions (arrows). (B) Two pairs of interfacing active sites (1–2 and 3–4) locate at opposite faces of the tetramer. SAXS profile (C) and ab-initio model (D) agree with the theoretical curve and high-resolution structure of the tetramer. (E) AUC assays show the presence of tetramers in solution, even at low protein concentrations.
Mentions: The two crystallographic structures of EaBglA, solved in distinct space groups, presented the same tetrameric arrangement (Fig. 2A). Structural comparisons revealed that the EaBglA subunits assemble in a different way from those of GH1 tetramers available at the Protein Data Bank (PDB), indicating that the tetrameric arrangement of EaBglA is the first of its kind to be reported within the GH1 family (Supplementary Fig. S2).

Bottom Line: Psychrophilic enzymes evolved from a plethora of structural scaffolds via multiple molecular pathways.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.The tetramer stabilizes the native conformation of the enzyme, leading to a 10-fold higher activity compared to the disassembled monomers.

View Article: PubMed Central - PubMed

Affiliation: Brazilian Bioethanol Science and Technology Laboratory, Campinas, São Paulo, Brazil.

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.

No MeSH data available.


Related in: MedlinePlus