Abstract
Β-glucosidases are key enzymes used in second-generation biofuel production. They act in the last step of the lignocellulose saccharification, converting cellobiose in glucose. However, most of the β-glucosidases are inhibited by high glucose concentrations, which turns it a limiting step for industrial production. Thus, β-glucosidases have been targeted by several studies aiming to understand the mechanism of glucose tolerance, pH and thermal resistance for constructing more efficient enzymes. In this paper, we present a database of β-glucosidase structures, called Glutantβase. Our database includes 3842 GH1 β-glucosidase sequences collected from UniProt. We modeled the sequences by comparison and predicted important features in the 3D-structure of each enzyme. Glutantβase provides information about catalytic and conserved amino acids, residues of the coevolution network, protein secondary structure, and residues located in the channel that guides to the active site. We also analyzed the impact of beneficial mutations reported in the literature, predicted in analogous positions, for similar enzymes. We suggested these mutations based on six previously described mutants that showed high catalytic activity, glucose tolerance, or thermostability (A404V, E96K, H184F, H228T, L441F, and V174C). Then, we used molecular docking to verify the impact of the suggested mutations in the affinity of protein and ligands (substrate and product). Our results suggest that only mutations based on the H228T mutant can reduce the affinity for glucose (product) and increase affinity for cellobiose (substrate), which indicates an increment in the resistance to product inhibition and agrees with computational and experimental results previously reported in the literature. More resistant β-glucosidases are essential to saccharification in industrial applications. However, thermostable and glucose-tolerant β-glucosidases are rare, and their glucose tolerance mechanisms appear to be related to multiple and complex factors. We gather here, a set of information, and made predictions aiming to provide a tool for supporting the rational design of more efficient β-glucosidases. We hope that Glutantβase can help improve second-generation biofuel production. Glutantβase is available at http://bioinfo.dcc.ufmg.br/glutantbase.
Highlights
Biofuels are a clean and renewable source of energy, rising as an alternative to fossil fuels, such as those derived from petroleum [1, 2]
We performed comparative modeling, predicted their secondary structure, detected the residues involved in coevolution networks, detailed the conserved residues, the catalytic glutamates, and the residues present in the substrate channel that guides to the active site
We modeled 5607 mutant proteins based on analogous positions of six beneficial mutations described in the literature: H228T [9], V174C [12], A404V [12], L441F [12], H184F [27], and E96K [47]
Summary
Biofuels are a clean and renewable source of energy, rising as an alternative to fossil fuels, such as those derived from petroleum [1, 2]. Yang et al [9] evaluated the importance of a set of amino acid positions through site-direct mutagenesis They reported that H228T and N301Q/V302F mutations could lead a marine non-resistant β-glucosidase to glucose tolerance. Betagdb uses the βglucosidase of the termite Neotermes koshunensis in complex to cellobiose (PDB ID: 3VIK) to detect the residues at 6.5 Å from any atom of the ligand (in this case, the substrate cellobiose) This value was defined based on the method proposed by [61] to construct representative fingerprints of protein pockets. For the β-glucosidase of a marine metagenome (UniProt ID: D0VEC8), the mutation H228T has been experimentally described as responsible for improving the glucose tolerance and, improving the β-glucosidase’s catalytic activity even in high concentrations of the product. We used BLAST [52] to perform searches for similar sequences inside Glutantβase
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