Molecular Dynamics Gives New Insights into the Glucose Tolerance and Inhibition Mechanisms on β-Glucosidases

Costa, Leon S. C.; Mariano, Diego; Rocha, Rafael E. O.; Kraml, Johannes; Silveira, Carlos H. da; Liedl, Klaus R.; Melo‐Minardi, Raquel Cardoso de; Lima, Leonardo Henrique França de

doi:10.3390/molecules24183215

Cited by 24 publications

(34 citation statements)

References 52 publications

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“…They act cleaving cellobiose in two molecules of glucose, which will be used in fermentation for producing bioethanol [3]. The design of thermostable and more efficient β-glucosidases enzymes has excellent value for the industry [4]; (ii) tobacco etch virus protease (PDB ID: 1LVB): proteases (peptidases) hydrolyze the peptide bond producing single amino acids or smaller polypeptides molecules. They are essential for several biological functions in all forms of life.…”

Section: Case Study 1: Evaluating Proteus Hypothesismentioning

confidence: 99%

“…The design of new proteins, known as protein engineering, has many applications for industry. Novel and likely enhanced proteins can benefit the development of new drugs, vaccines, treatment therapies, and improve the enzymes used in digestive processes for new food and biofuel production [1][2][3][4]. Industrial biotechnological applications require enzymes with higher thermal and conformational stability [5].…”

Section: Introductionmentioning

confidence: 99%

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Proteus: An algorithm for proposing stabilizing mutation pairs based on interactions observed in known protein 3D structures

et al. 2020

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Background: Protein engineering has many applications for industry, such as the development of new drugs, vaccines, treatment therapies, food, and biofuel production. A common way to engineer a protein is to perform mutations in functionally essential residues to optimize their function. However, the discovery of beneficial mutations for proteins is a complex task, with a time-consuming and high cost for experimental validation. Hence, computational approaches have been used to propose new insights for experiments narrowing the search space and reducing the costs. Results: In this study, we developed Proteus (an acronym for Protein Engineering Supporter), a new algorithm for proposing mutation pairs in a target 3D structure. These suggestions are based on contacts observed in other known structures from Protein Data Bank (PDB). Proteus' basic assumption is that if a non-interacting pair of amino acid residues in the target structure is exchanged to an interacting pair, this could enhance protein stability. This trade is only allowed if the main-chain conformation of the residues involved in the contact is conserved. Furthermore, no steric impediment is expected between the proposed mutations and the surrounding protein atoms. To evaluate Proteus, we performed two case studies with proteins of industrial interests. In the first case study, we evaluated if the mutations suggested by Proteus for four protein structures enhance the number of inter-residue contacts. Our results suggest that most mutations proposed by Proteus increase the number of interactions into the protein. In the second case study, we used Proteus to suggest mutations for a lysozyme protein. Then, we compared Proteus' outcomes to mutations with available experimental evidence reported in the ProTherm database. Four mutations, in which our results agree with the experimental data, were found. This could be initial evidence that changes in the side-chain of some residues do not cause disturbances that harm protein structure stability.

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Section: Case Study 1: Evaluating Proteus Hypothesismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Proteus: An algorithm for proposing stabilizing mutation pairs based on interactions observed in known protein 3D structures

et al. 2020

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“…3.2.1.21) are enzymes that act in the last step of the saccharification process, cleaving cellobiose into two molecules of glucose 28,29 . Hence, they are considered very important for the second-generation biofuel industrial applications [30][31][32] . Besides, the literature has reported that most β-glucosidases are inhibited in high glucose concentrations 33,34 .…”

Section: Sars -Cov R1-r2mentioning

confidence: 99%

“…Overall, minimal topological modifications represent a promising strategy for suggesting mutations, especially in the beta-glucosidase loop regions that surround the active site. The topology and dynamics of these loops can allow or restrict movements involved in glucose entrance and exit (i.e., glucose tolerance) 30,32,36,43 or also can affect the thermostability [41][42][43] . These are both examples of industrially desirable characteristics for these and other proteins.…”

Section: Implications For Protein Engineeringmentioning

confidence: 99%

VTR: an algorithm for identifying analogous contacts on protein structures and their complexes

Pimentel

Mariano

Cantão

et al. 2020

Preprint

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Evolutionarily related proteins can present similar structures but very dissimilar sequences. Hence, understanding the role of the inter-residues contacts for the protein structure has been the target of many studies. Contacts comprise non-covalent interactions, which are essential to stabilize macromolecular structures such as proteins. Here we show VTR, a new method for the detection of analogous contacts in protein pairs. VTR performs structural alignment between proteins and detects interactions that occur in similar regions. To evaluate our tool, we proposed three case studies: (i) we compared a vertebrate myoglobin and a truncated invertebrate hemoglobin; (ii) analyzed interactions between the spike protein RBD of SARS-CoV-2 and the cell receptor ACE2; and (iii) compared a glucose-tolerant and a non-tolerant β-glucosidase enzyme used for biofuel production. The case studies demonstrate the potential of VTR for the understanding of functional similarities between distantly sequence-related proteins, as well as the exploration of important drug targets and rational design of enzymes for industrial applications. We envision VTR as a promising tool for understanding differences and similarities between homologous proteins with similar 3D structures but different sequences. VTR is available at http://bioinfo.dcc.ufmg.br/vtr.

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“…From these 15 proposed mutations, a previous study has provided experimental evidence of enhancing β-glucosidase activity even in high glucose concentrations for three of them: H228C, H228T, and H228V [9]. The residues mutated V302F, N301Q/V302F, F172I, V227M, G246S, T299S, and H228T were also the target of other computational studies that used classic and accelerated molecular dynamics simulation to highlight their role in glucose releasing [45,46]. Despite all these efforts, the rational design of more efficient βglucosidases is still a challenge.…”

Section: Introductionmentioning

confidence: 99%

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

Mariano

Pantuza

Santos

et al. 2020

BMC Mol and Cell Biol

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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.

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Molecular Dynamics Gives New Insights into the Glucose Tolerance and Inhibition Mechanisms on β-Glucosidases

Cited by 24 publications

References 52 publications

Proteus: An algorithm for proposing stabilizing mutation pairs based on interactions observed in known protein 3D structures

Proteus: An algorithm for proposing stabilizing mutation pairs based on interactions observed in known protein 3D structures

VTR: an algorithm for identifying analogous contacts on protein structures and their complexes

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

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