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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

Structure and sequence analyses of the cold-adapted EaBglA.(A) Cartoon representation of EaBglA superimposed on the thermophilic HoBglA, showing the catalytic residues as sticks. (B) Fraction of non-conservative substitutions referent to non-polar, neutral polar or charged residues from the enzymes DAU5-BglA and HoBglA replaced by residues of a different category (X) in EaBglA. (C) Residues specific to the cold-adapted enzyme (according to a sequence alignment between EaBglA, DAU5-BglA and HoBglA) mapped in the 3D structure and colored according to its solvent accessibility from yellow (buried) to blue (fully exposed). Catalytic glutamates are shown in red. (D) Number of residues specific to the cold-adapted enzyme classified according to their localization in the protein secondary structure (top chart) and solvent accessibility (bottom chart).
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f1: Structure and sequence analyses of the cold-adapted EaBglA.(A) Cartoon representation of EaBglA superimposed on the thermophilic HoBglA, showing the catalytic residues as sticks. (B) Fraction of non-conservative substitutions referent to non-polar, neutral polar or charged residues from the enzymes DAU5-BglA and HoBglA replaced by residues of a different category (X) in EaBglA. (C) Residues specific to the cold-adapted enzyme (according to a sequence alignment between EaBglA, DAU5-BglA and HoBglA) mapped in the 3D structure and colored according to its solvent accessibility from yellow (buried) to blue (fully exposed). Catalytic glutamates are shown in red. (D) Number of residues specific to the cold-adapted enzyme classified according to their localization in the protein secondary structure (top chart) and solvent accessibility (bottom chart).

Mentions: Crystallographic studies showed that the (α/β)8-barrel fold and the catalytic residues Glu162 (acid/base) and Glu350 (nucleophile), typical of GH1 members1920, are conserved in EaBglA (Fig. 1A). Interestingly, the cold-adapted EaBglA is closely related to a thermostable β-glucosidase from Halothermotrix orenii (HoBglA)21 (Fig. 1A), sharing 48% of sequence identity that, as expected, reflects in a high structural similarity with only small main-chain displacements in peripheral loops and α-helices (Table 1, Fig. 1A).


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)

Structure and sequence analyses of the cold-adapted EaBglA.(A) Cartoon representation of EaBglA superimposed on the thermophilic HoBglA, showing the catalytic residues as sticks. (B) Fraction of non-conservative substitutions referent to non-polar, neutral polar or charged residues from the enzymes DAU5-BglA and HoBglA replaced by residues of a different category (X) in EaBglA. (C) Residues specific to the cold-adapted enzyme (according to a sequence alignment between EaBglA, DAU5-BglA and HoBglA) mapped in the 3D structure and colored according to its solvent accessibility from yellow (buried) to blue (fully exposed). Catalytic glutamates are shown in red. (D) Number of residues specific to the cold-adapted enzyme classified according to their localization in the protein secondary structure (top chart) and solvent accessibility (bottom chart).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Structure and sequence analyses of the cold-adapted EaBglA.(A) Cartoon representation of EaBglA superimposed on the thermophilic HoBglA, showing the catalytic residues as sticks. (B) Fraction of non-conservative substitutions referent to non-polar, neutral polar or charged residues from the enzymes DAU5-BglA and HoBglA replaced by residues of a different category (X) in EaBglA. (C) Residues specific to the cold-adapted enzyme (according to a sequence alignment between EaBglA, DAU5-BglA and HoBglA) mapped in the 3D structure and colored according to its solvent accessibility from yellow (buried) to blue (fully exposed). Catalytic glutamates are shown in red. (D) Number of residues specific to the cold-adapted enzyme classified according to their localization in the protein secondary structure (top chart) and solvent accessibility (bottom chart).
Mentions: Crystallographic studies showed that the (α/β)8-barrel fold and the catalytic residues Glu162 (acid/base) and Glu350 (nucleophile), typical of GH1 members1920, are conserved in EaBglA (Fig. 1A). Interestingly, the cold-adapted EaBglA is closely related to a thermostable β-glucosidase from Halothermotrix orenii (HoBglA)21 (Fig. 1A), sharing 48% of sequence identity that, as expected, reflects in a high structural similarity with only small main-chain displacements in peripheral loops and α-helices (Table 1, Fig. 1A).

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