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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

Phylogenetic tree of GH1 β-glucosidases in bacteria.Unrooted tree of 44 protein sequences of GH1 β-glucosidases as inferred by the ML method using the MEGA657 program. Numbers at nodes represent bootstrap values from 1000 replications (shown as percentage and only those higher than 50%). Scale bar indicates estimated number of substitutions per site. Taxonomic classification at the phylum level (Actinobacteria, Firmicutes and Proteobacteria) are indicated, subdividing the Proteobacteria phylum into the two classes represented in the tree (α and γ). Colored circles indicate the classification of each specie as psychrophile/psychrotroph (blue), mesophile (light orange), and thermophile (red) according to literature data (see Table S5). Cyan box indicates the E. antarcticum position in the tree.
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f7: Phylogenetic tree of GH1 β-glucosidases in bacteria.Unrooted tree of 44 protein sequences of GH1 β-glucosidases as inferred by the ML method using the MEGA657 program. Numbers at nodes represent bootstrap values from 1000 replications (shown as percentage and only those higher than 50%). Scale bar indicates estimated number of substitutions per site. Taxonomic classification at the phylum level (Actinobacteria, Firmicutes and Proteobacteria) are indicated, subdividing the Proteobacteria phylum into the two classes represented in the tree (α and γ). Colored circles indicate the classification of each specie as psychrophile/psychrotroph (blue), mesophile (light orange), and thermophile (red) according to literature data (see Table S5). Cyan box indicates the E. antarcticum position in the tree.

Mentions: To evaluate if the strategy that rendered EaBglA cold active is conserved in the GH1 family, we constructed a phylogenetic tree based on protein sequences of GH1 β-glucosidases from psychrotrophic/psychrophilic bacteria and their closest relatives (Fig. 7). This analysis revealed that cold-adapted β-glucosidases have emerged several times during the evolution of bacteria and in different phyla such as Firmicutes, Actinobacteria and Proteobacteria (Fig. 7). Therefore, the set of molecular modifications selected by nature to produce cold-active β-glucosidases might be different in these distantly related enzymes. Supporting this hypothesis, multiple sequence alignment shows that the residues forming the tetrameric interfaces of EaBglA are poorly conserved in other enzymes from psychro-tolerant bacteria, suggesting that this particular oligomerization strategy emerged specifically in the Exiguobacterium clade (Supplementary Fig. S3).


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)

Phylogenetic tree of GH1 β-glucosidases in bacteria.Unrooted tree of 44 protein sequences of GH1 β-glucosidases as inferred by the ML method using the MEGA657 program. Numbers at nodes represent bootstrap values from 1000 replications (shown as percentage and only those higher than 50%). Scale bar indicates estimated number of substitutions per site. Taxonomic classification at the phylum level (Actinobacteria, Firmicutes and Proteobacteria) are indicated, subdividing the Proteobacteria phylum into the two classes represented in the tree (α and γ). Colored circles indicate the classification of each specie as psychrophile/psychrotroph (blue), mesophile (light orange), and thermophile (red) according to literature data (see Table S5). Cyan box indicates the E. antarcticum position in the tree.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Phylogenetic tree of GH1 β-glucosidases in bacteria.Unrooted tree of 44 protein sequences of GH1 β-glucosidases as inferred by the ML method using the MEGA657 program. Numbers at nodes represent bootstrap values from 1000 replications (shown as percentage and only those higher than 50%). Scale bar indicates estimated number of substitutions per site. Taxonomic classification at the phylum level (Actinobacteria, Firmicutes and Proteobacteria) are indicated, subdividing the Proteobacteria phylum into the two classes represented in the tree (α and γ). Colored circles indicate the classification of each specie as psychrophile/psychrotroph (blue), mesophile (light orange), and thermophile (red) according to literature data (see Table S5). Cyan box indicates the E. antarcticum position in the tree.
Mentions: To evaluate if the strategy that rendered EaBglA cold active is conserved in the GH1 family, we constructed a phylogenetic tree based on protein sequences of GH1 β-glucosidases from psychrotrophic/psychrophilic bacteria and their closest relatives (Fig. 7). This analysis revealed that cold-adapted β-glucosidases have emerged several times during the evolution of bacteria and in different phyla such as Firmicutes, Actinobacteria and Proteobacteria (Fig. 7). Therefore, the set of molecular modifications selected by nature to produce cold-active β-glucosidases might be different in these distantly related enzymes. Supporting this hypothesis, multiple sequence alignment shows that the residues forming the tetrameric interfaces of EaBglA are poorly conserved in other enzymes from psychro-tolerant bacteria, suggesting that this particular oligomerization strategy emerged specifically in the Exiguobacterium clade (Supplementary Fig. S3).

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