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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 tetramer promotes local changes in flexibility.(A) Comparison of the backbone root mean square fluctuations (RMSF) of EaBglA tetramer chains (TA-TD) and the HoBglA monomer at 10 °C. For comparison purposes, the RMSF profiles were aligned according to the structural alignment between EaBglA and HoBglA (assuming EaBglA numbering as reference). The position of the catalytic residues (asterisk) and the β10/α16 loop (triangle) are indicated. (B) Surface representation of an EaBglA subunit highlighting the catalytic residues (red), the tetramer interfaces (α and γ) and the residues with higher flexibility (dark cyan) compared to HoBglA (ΔRMSF > 0.25 Å in at least two tetramer subunits). Protein views correspond to Fig. 1C. N- and C-termini as well as the β10/α16 loop are indicated. (C) N- and C-termini of HoBglA (orange) superimposed onto EaBglA (colored according to panel B). Dashed lines represent main-chain hydrogen bonds connecting N- and C-terminal residues (sticks) in the thermophilic enzyme. (D) The solvent-exposed residues of β10/α16 loop (dark cyan sticks) are more flexible in the cold-adapted enzyme. Some of them are specific to EaBglA compared to HoBglA and Dau5-BglA (asterisk). The β10/α16 loop are near Phe412. The latter along with Glu403, Gln17 and Asn161 delineate the -1 subsite and display higher RMSF values than their HoBglA counterparts do in most tetramer subunits, according to the MD simulation at 10 °C. Red labels indicate catalytic residues.
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f5: The tetramer promotes local changes in flexibility.(A) Comparison of the backbone root mean square fluctuations (RMSF) of EaBglA tetramer chains (TA-TD) and the HoBglA monomer at 10 °C. For comparison purposes, the RMSF profiles were aligned according to the structural alignment between EaBglA and HoBglA (assuming EaBglA numbering as reference). The position of the catalytic residues (asterisk) and the β10/α16 loop (triangle) are indicated. (B) Surface representation of an EaBglA subunit highlighting the catalytic residues (red), the tetramer interfaces (α and γ) and the residues with higher flexibility (dark cyan) compared to HoBglA (ΔRMSF > 0.25 Å in at least two tetramer subunits). Protein views correspond to Fig. 1C. N- and C-termini as well as the β10/α16 loop are indicated. (C) N- and C-termini of HoBglA (orange) superimposed onto EaBglA (colored according to panel B). Dashed lines represent main-chain hydrogen bonds connecting N- and C-terminal residues (sticks) in the thermophilic enzyme. (D) The solvent-exposed residues of β10/α16 loop (dark cyan sticks) are more flexible in the cold-adapted enzyme. Some of them are specific to EaBglA compared to HoBglA and Dau5-BglA (asterisk). The β10/α16 loop are near Phe412. The latter along with Glu403, Gln17 and Asn161 delineate the -1 subsite and display higher RMSF values than their HoBglA counterparts do in most tetramer subunits, according to the MD simulation at 10 °C. Red labels indicate catalytic residues.

Mentions: To gather insight into the dynamic properties of the tetramer and its potential role in cold adaptation, we performed molecular dynamics simulations using the biological units of EaBglA (tetramer) and HoBglA (monomer)21. The two proteins have similar overall flexibility, but with local changes that might affect enzymatic activity (Table 2, Fig. 5A).


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 tetramer promotes local changes in flexibility.(A) Comparison of the backbone root mean square fluctuations (RMSF) of EaBglA tetramer chains (TA-TD) and the HoBglA monomer at 10 °C. For comparison purposes, the RMSF profiles were aligned according to the structural alignment between EaBglA and HoBglA (assuming EaBglA numbering as reference). The position of the catalytic residues (asterisk) and the β10/α16 loop (triangle) are indicated. (B) Surface representation of an EaBglA subunit highlighting the catalytic residues (red), the tetramer interfaces (α and γ) and the residues with higher flexibility (dark cyan) compared to HoBglA (ΔRMSF > 0.25 Å in at least two tetramer subunits). Protein views correspond to Fig. 1C. N- and C-termini as well as the β10/α16 loop are indicated. (C) N- and C-termini of HoBglA (orange) superimposed onto EaBglA (colored according to panel B). Dashed lines represent main-chain hydrogen bonds connecting N- and C-terminal residues (sticks) in the thermophilic enzyme. (D) The solvent-exposed residues of β10/α16 loop (dark cyan sticks) are more flexible in the cold-adapted enzyme. Some of them are specific to EaBglA compared to HoBglA and Dau5-BglA (asterisk). The β10/α16 loop are near Phe412. The latter along with Glu403, Gln17 and Asn161 delineate the -1 subsite and display higher RMSF values than their HoBglA counterparts do in most tetramer subunits, according to the MD simulation at 10 °C. Red labels indicate catalytic residues.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4815018&req=5

f5: The tetramer promotes local changes in flexibility.(A) Comparison of the backbone root mean square fluctuations (RMSF) of EaBglA tetramer chains (TA-TD) and the HoBglA monomer at 10 °C. For comparison purposes, the RMSF profiles were aligned according to the structural alignment between EaBglA and HoBglA (assuming EaBglA numbering as reference). The position of the catalytic residues (asterisk) and the β10/α16 loop (triangle) are indicated. (B) Surface representation of an EaBglA subunit highlighting the catalytic residues (red), the tetramer interfaces (α and γ) and the residues with higher flexibility (dark cyan) compared to HoBglA (ΔRMSF > 0.25 Å in at least two tetramer subunits). Protein views correspond to Fig. 1C. N- and C-termini as well as the β10/α16 loop are indicated. (C) N- and C-termini of HoBglA (orange) superimposed onto EaBglA (colored according to panel B). Dashed lines represent main-chain hydrogen bonds connecting N- and C-terminal residues (sticks) in the thermophilic enzyme. (D) The solvent-exposed residues of β10/α16 loop (dark cyan sticks) are more flexible in the cold-adapted enzyme. Some of them are specific to EaBglA compared to HoBglA and Dau5-BglA (asterisk). The β10/α16 loop are near Phe412. The latter along with Glu403, Gln17 and Asn161 delineate the -1 subsite and display higher RMSF values than their HoBglA counterparts do in most tetramer subunits, according to the MD simulation at 10 °C. Red labels indicate catalytic residues.
Mentions: To gather insight into the dynamic properties of the tetramer and its potential role in cold adaptation, we performed molecular dynamics simulations using the biological units of EaBglA (tetramer) and HoBglA (monomer)21. The two proteins have similar overall flexibility, but with local changes that might affect enzymatic activity (Table 2, Fig. 5A).

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