Glutantβase: a database for improving the rational design of glucose-tolerant β-glucosidases

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 βglucosidases 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.

Structural determinants of cold activity and glucose tolerance of a family 1 glycoside hydrolase (GH1) from Antarctic Marinomonas sp. ef1

“…Although the mechanism of glucose stimulation/inhibition remains elusive, structural analyses and literature data on glucose‐tolerant GH1s allowed us to identify the residues likely involved in M‐GH1 glucose tolerance [30]. In particular, we focused on the comparison of M‐GH1 with the glucose‐stimulated β‐glucosidase from Humicola insolens (HiBG, PDB: 4MDP [31]), which share 39.9% of sequence identity and reasonable structural homology (RMSD ~1.7 Å over 419 aligned residues, Fig.…”

“…glucose tolerance [30]. In particular, we focused on the comparison of M-GH1 with the glucosestimulated β-glucosidase from Humicola insolens (HiBG, PDB: 4MDP [31]), which share 39.9% of sequence identity and reasonable structural homology (RMSD $1.7 Å over 419 aligned residues, Fig.…”

VTR: A Web Tool for Identifying Analogous Contacts on Protein Structures and Their Complexes

“…For the third case study, we selected the sequences of the glucose-tolerant GH1 β-glucosidase of a South China Sea metagenome (Bgl1A; UniProt ID: D5KX75) ( Fang et al, 2010 ) and the non-tolerant GH1 β-glucosidase of a South China Sea metagenome (Bgl1B; UniProt ID: D0VEC8) ( Fang et al, 2009 ) from Glutantbase ( Mariano et al, 2020 ). We also constructed two mutants, H57D (Bgl1A) and D57H (Bgl1B), to evaluate VTR’s ability to propose mutations for enzymes based on differences of contacts ( Supplementary Tables S5–S6 ).…”

Multidisciplinary approaches for enzyme biocatalysis in pharmaceuticals: protein engineering, computational biology, and nanoarchitectonics

“…Further exemplification of enzyme engineering can be found in Table 2, where additional instances are provided. [122][123][124][125][126][127][128][129][130][131][132][133][134][135][136][137][138][139][140] Enhanced catalytic activity accelerates reactions, stability ensures that enzymes can endure challenging environments, and refined substrate specificity opens doors to a plethora of novel applications. However, these methods still have several limitations, for example, directed evolution can be timeconsuming and resource-intensive and may not always yield enzymes with the desired properties.…”