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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 interfaces of EaBglA tetramer.(A) The interface α involves residues from the α-helices α11 and α13, while the interface γ connects loops from the catalytic face. (B) Two hydrophobic clusters (labeled residues) with a two-fold symmetry stabilize the interface α along with few hydrogen bonds (dotted lines) between interfacing polar residues (sticks). (C) The interface γ is essentially linked by hydrogen bonds (dotted lines) and electrostatic interactions (Lys41-Glu300), forming a semi-circle (light blue) that connects two active sites.
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f3: The interfaces of EaBglA tetramer.(A) The interface α involves residues from the α-helices α11 and α13, while the interface γ connects loops from the catalytic face. (B) Two hydrophobic clusters (labeled residues) with a two-fold symmetry stabilize the interface α along with few hydrogen bonds (dotted lines) between interfacing polar residues (sticks). (C) The interface γ is essentially linked by hydrogen bonds (dotted lines) and electrostatic interactions (Lys41-Glu300), forming a semi-circle (light blue) that connects two active sites.

Mentions: The tetramer has a 222 symmetry (with a distorted tetrahedral geometry) and two pairs of active sites placed at opposite faces (Fig. 2A,B). Each pair shares the same entrance and forms a huge cavity [11393 Å3 ± 216 (mean ± SD)] with a two-fold symmetry (Fig. 2B). Two types of interface stabilize the EaBglA tetramer (called here α and γ). The type α is a side-to-side interface composed by residues from the helices α11 and α13 and the loops α13/α14 and β8/β9 (Fig. 3A). It comprises two hydrophobic clusters surrounded by few hydrogen bonds (Fig. 3B). The γ interface forms a semi-circle wall at the catalytic face of the TIM barrel, connecting the active sites from neighboring subunits (Fig. 3C,D). It comprises polar residues involved in the formation of several hydrogen bonds (Fig. 3D).


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 interfaces of EaBglA tetramer.(A) The interface α involves residues from the α-helices α11 and α13, while the interface γ connects loops from the catalytic face. (B) Two hydrophobic clusters (labeled residues) with a two-fold symmetry stabilize the interface α along with few hydrogen bonds (dotted lines) between interfacing polar residues (sticks). (C) The interface γ is essentially linked by hydrogen bonds (dotted lines) and electrostatic interactions (Lys41-Glu300), forming a semi-circle (light blue) that connects two active sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: The interfaces of EaBglA tetramer.(A) The interface α involves residues from the α-helices α11 and α13, while the interface γ connects loops from the catalytic face. (B) Two hydrophobic clusters (labeled residues) with a two-fold symmetry stabilize the interface α along with few hydrogen bonds (dotted lines) between interfacing polar residues (sticks). (C) The interface γ is essentially linked by hydrogen bonds (dotted lines) and electrostatic interactions (Lys41-Glu300), forming a semi-circle (light blue) that connects two active sites.
Mentions: The tetramer has a 222 symmetry (with a distorted tetrahedral geometry) and two pairs of active sites placed at opposite faces (Fig. 2A,B). Each pair shares the same entrance and forms a huge cavity [11393 Å3 ± 216 (mean ± SD)] with a two-fold symmetry (Fig. 2B). Two types of interface stabilize the EaBglA tetramer (called here α and γ). The type α is a side-to-side interface composed by residues from the helices α11 and α13 and the loops α13/α14 and β8/β9 (Fig. 3A). It comprises two hydrophobic clusters surrounded by few hydrogen bonds (Fig. 3B). The γ interface forms a semi-circle wall at the catalytic face of the TIM barrel, connecting the active sites from neighboring subunits (Fig. 3C,D). It comprises polar residues involved in the formation of several hydrogen bonds (Fig. 3D).

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