RESUMO
Recently, a bacterium strain of Ideonella sakaiensis was identified with the uncommon ability to degrade the poly(ethylene terephthalate) (PET). The PETase from I. sakaiensis strain 201-F6 (IsPETase) catalyzes the hydrolysis of PET converting it to mono(2-hydroxyethyl) terephthalic acid (MHET), bis(2-hydroxyethyl)-TPA (BHET), and terephthalic acid (TPA). Despite the potential of this enzyme for mitigation or elimination of environmental contaminants, one of the limitations of the use of IsPETase for PET degradation is the fact that it acts only at moderate temperature due to its low thermal stability. Besides, molecular details of the main interactions of PET in the active site of IsPETase remain unclear. Herein, molecular docking and molecular dynamics (MD) simulations were applied to analyze structural changes of IsPETase induced by PET binding. Results from the essential dynamics revealed that the ß1-ß2 connecting loop is very flexible. This loop is located far from the active site of IsPETase and we suggest that it can be considered for mutagenesis to increase the thermal stability of IsPETase. The free energy landscape (FEL) demonstrates that the main change in the transition between the unbound to the bound state is associated with the ß7-α5 connecting loop, where the catalytic residue Asp206 is located. Overall, the present study provides insights into the molecular binding mechanism of PET into the IsPETase structure and a computational strategy for mapping flexible regions of this enzyme, which can be useful for the engineering of more efficient enzymes for recycling plastic polymers using biological systems.
Assuntos
Proteínas de Bactérias/metabolismo , Burkholderiales/metabolismo , Hidrolases/metabolismo , Polietilenotereftalatos/metabolismo , Biocatálise , HidróliseRESUMO
Chagas' disease is considered to be a health problem affecting millions of people in Latin America. This disease is caused by the parasite Trypanosoma cruzi. Recently dihydroorotate dehydrogenase class 1A from Trypanosoma cruzi (TcDHODA) was shown to be essential for the survival and growth of T. cruzi and proposed as a drug target against Chagas' disease. This enzyme catalyzes the oxidation of (S)-dihydroorotate to orotate, with a proposed catalytic cycle consisting of two half-reactions. In the first half-reaction dihydroorotate is oxidized to orotate, with the consequent reduction of the flavin mononucleotide cofactor. In the second half-reaction fumarate is reduced to succinate. The first oxidation half-reaction may occur via a concerted or a stepwise mechanism. Herein, the catalytic mechanism of TcDHODA has been studied using hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) Molecular Dynamics (MD) simulations. The free energy profiles derived from the bidimensional potential of mean force reveal more details for two half-reaction processes.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Trypanosoma cruzi/enzimologia , Biocatálise , Di-Hidro-Orotato Desidrogenase , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Ácido Orótico/análogos & derivados , Ácido Orótico/química , Ácido Orótico/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Teoria Quântica , Eletricidade EstáticaRESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been identified as a key enzyme involved in glycolysis processes for energy production in the Trypanosoma cruzi parasite. This enzyme catalyses the oxidative phosphorylation of glyceraldehyde 3-phosphate (G3P) in the presence of inorganic phosphate (Pi) and nicotinamide adenosine dinucleotide (NAD+). The catalytic mechanism used by GAPDH has been intensively investigated. However, the individual roles of Pi and the C3 phosphate of G3P (Ps) sites, as well as some residues such as His194 in the catalytic mechanism, remain unclear. In this study, we have employed Molecular Dynamics (MD) simulations within hybrid quantum mechanical/molecular mechanical (QM/MM) potentials to obtain the Potential of Mean Force of the catalytic oxidative phosphorylation mechanism of the G3P substrate used by GAPDH. According to our results, the first stage of the reaction (oxidoreduction) takes place in the Pi site (energetically more favourable), with the formation of oxyanion thiohemiacetal and thioacylenzyme intermediates without acid-base assistance of His194. Analysis of the interaction energy by residues shows that Arg249 has an important role in the ability of the enzyme to bind the G3P substrate, which interacts with NAD+ and other important residues, such as Cys166, Glu109, Thr167, Ser247 and Thr226, in the GAPDH active site. Finally, the inhibition mechanism of the GAPDH enzyme by the 3-(p-nitrophenoxycarboxyl)-3-ethylene propyl dihydroxyphosphonate inhibitor was investigated in order to contribute to the design of new inhibitors of GAPDH from Trypanosoma cruzi.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Simulação de Dinâmica Molecular , Teoria Quântica , Trypanosoma cruzi/enzimologia , Biocatálise , Gliceraldeído-3-Fosfato Desidrogenases/química , NAD/química , NAD/metabolismo , Oxirredução , Fosforilação Oxidativa , Especificidade por SubstratoRESUMO
The substitution of serine and threonine residues in nucleocytoplasmic proteins with 2-acetamido-2-deoxy-ß-D-glucopyranose (O-GlcNAc) residues is an essential post-translational modification found in many multicellular eukaryotes. O-glycoprotein 2-acetamino-2-deoxy-ß-D-glucopyranosidase (O-GlcNAcase) hydrolyzes O-GlcNAc residues from post-translationally modified serine/threonine residues of nucleocytoplasmic protein. O-GlcNAc has been implicated in several disease states such as cancer, Alzheimer's disease, and type II diabetes. For this paper, a model of the human O-GlcNAcase (hOGA) enzyme based on the X-ray structures of bacterial Clostridium perfringens (CpNagJ) and Bacteroides thetaiotaomicrometer (BtOGA) homologues has been generated through molecular homology modeling. In addition, molecular docking, molecular dynamics (MD) simulations, and Linear Interaction Energy (LIE) were employed to determine the bind for derivatives of two potent inhibitors: O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2'-methyl-R-D-glucopyranoso-[2,1-d]-Δ2'-thiazoline (NAG-thiazoline), with hOGA. The results show that the binding free energy calculations using the Linear Interaction Energy (LIE) are correlated with inhibition constant values. Therefore, the model of the human O-GlcNAcase (hOGA) obtained here may be used as a target for rational design of new inhibitors.
Assuntos
Acetilglucosamina/análogos & derivados , Proteínas de Bactérias/química , Simulação de Acoplamento Molecular , Oximas/química , Fenilcarbamatos/química , Tiazóis/química , beta-N-Acetil-Hexosaminidases/química , Acetilglucosamina/química , Proteínas de Bactérias/antagonistas & inibidores , Bacteroides/química , Bacteroides/enzimologia , Sítios de Ligação , Clostridium perfringens/química , Clostridium perfringens/enzimologia , Cristalografia por Raios X , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Cinética , Ligantes , Ligação Proteica , Conformação Proteica , Homologia Estrutural de Proteína , Termodinâmica , beta-N-Acetil-Hexosaminidases/antagonistas & inibidoresRESUMO
O-Glycoprotein 2-acetamino-2-deoxy-ß-D-glucopyranosidase (O-GlcNAcase) hydrolyzes O-linked 2-acetamido-2-deoxy-ß-D-glucopyranoside (O-GlcNAc) residues from post-translationally modified serine/threonine residues of nucleocytoplasmic protein. The chemical process involves substrate-assisted catalysis, where two aspartate residues have been identified as the two key catalytic residues of O-GlcNAcase. In this report, the first step of the catalytic mechanism used by O-GlcNAcase involving substrate-assisted catalysis has been studied using a hybrid quantum mechanical/molecular mechanical (QM/MM) Molecular Dynamics (MD) calculations. The free energy profile shows that the formation of the oxazoline intermediate in the O-GlcNAcase catalytic reaction takes place by means of a stepwise mechanism. The first step would be a cyclization of the acetomide group, which seems to be dependent on the proton transfer from a conserved aspartate, Asp298 in Clostridium perfringens O-GlcNAcase. From this new intermediate, a proton is transferred from the azoline ring to another conserved aspartate (Asp297) thus forming the oxazoline ion and departure of the aglycone. In addition, averaged values of protein-substrate interaction energy along the reaction path shows that, in fact, the transition states present the highest binding affinities. A deeper analysis of the binding contribution of the individual residues shows that Asp297, Asp298, and Asp401 are basically responsible of the stabilization of these complexes. These results would explain why O-(2-acetamido-2deoxy-d-glucopyranosylidene)amino-N-phenycarbamate (PUGNAc), 1,2-dideoxy-2'-methyl-α-D-glucopyranoso-[2,1-d]-Δ2'-thiazoline (NAG-thiazoline), and GlcNAcstatin derivatives are potent inhibitors of this enzyme, resembling the two transition states of the O-GlcNAcase catalytic reaction path. These results may be useful to rational design compounds with more interesting inhibitory activity.
Assuntos
Clostridium perfringens/enzimologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismo , Clostridium perfringens/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Simulação de Dinâmica Molecular , Oxazóis/metabolismo , Teoria Quântica , Especificidade por Substrato , Termodinâmica , beta-N-Acetil-Hexosaminidases/químicaRESUMO
The enzyme O-glycoprotein 2-acetamino-2-deoxy-beta-d-glucopyranosidase (O-GlcNAcase) is responsible for the removal of N-acetylglucosamine moieties from 2-acetamido-2-deoxy-beta-D-glucopyranose (O-GlcNAc) residues of serine/threonine residues of modified proteins. We herein present results of hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations applied to the study of the interactions established between a bacterial Clostridium perfringens homologue (CpNagJ) and PUGNAc, a potent known inhibitor of this enzyme. Electrostatic binding free energy and energy term decomposition have been computed for the wild-type CpNagJ and several mutants: D297N, D298N, Y335F, N390A, N396A, D401A, and W490A. The theoretical results have been compared with recently experimental data based on crystallographic and mutation studies on the same system. Our results reveal that, first, there is a strong interaction between Asp401, Asp298, and Asp297 residues and the PUGNAc inhibitor; and, second, the electrostatic substrate binding free energy is higher in wild-type, N390A and W490A mutants than in D297N, D298N, Y335F, N396A, and D401A ones, in accordance with the experimental results. Finally, both our theoretical predictions and the experimental data are compatible with a substrate-assisted reaction mechanism, involving two conserved aspartate residues.
Assuntos
Acetilglucosamina/análogos & derivados , Clostridium perfringens/enzimologia , Simulação de Dinâmica Molecular , Oximas/química , Fenilcarbamatos/química , beta-N-Acetil-Hexosaminidases/química , Acetilglucosamina/química , Substituição de Aminoácidos , Cristalografia por Raios X , Mutagênese Sítio-Dirigida , Ligação Proteica , Teoria Quântica , Eletricidade Estática , Termodinâmica , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
O-glycoprotein 2-acetamino-2-deoxy-beta- d-glucopyranosidase ( O-GlcNAcase) hydrolyzes 2-acetamido-2-deoxy-beta- d-glucopyranose ( O-GlcNAc) residues of serine/threonine residues of modified proteins. O-GlcNAc is present in many intracellular proteins and appears to have a role in the etiology of several diseases including cancer, Alzheimer's disease, and type II diabetes. In this work, we have carried out molecular dynamics simulations using a hybrid quantum mechanics/molecular mechanics approach to determine the binding of two potent inhibitors, PUGNAc and NAG, with a bacterial O-GlcNAcase. The results of these simulations show that Asp-401, Asp-298, and Asp-297 residues play an important role in the protein-inhibitor interactions. These results might be useful to design compounds with more interesting inhibitory activity on the basis of its three-dimensional structure.
Assuntos
Acetilglucosamina/análogos & derivados , Inibidores Enzimáticos/química , Modelos Moleculares , Oximas/química , Fenilcarbamatos/química , Teoria Quântica , Tiazóis/química , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/química , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Acetilglucosamina/farmacologia , Sítios de Ligação , Biocatálise/efeitos dos fármacos , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Ligantes , Conformação Molecular , Oximas/metabolismo , Oximas/farmacologia , Fenilcarbamatos/metabolismo , Fenilcarbamatos/farmacologia , Ligação Proteica , Prótons , Eletricidade Estática , Termodinâmica , Tiazóis/metabolismo , Tiazóis/farmacologia , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Integrase (IN) is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective viral replication. Recently, mutation studies have been reported that have shown that a certain degree of viral resistance to diketo acids (DKAs) appears when some amino acid residues of the IN active site are mutated. Mutations represent a fascinating experimental challenge, and we invite theoretical simulations for the disclosure of still unexplored features of enzyme reactions. The aim of this work is to understand the molecular mechanisms of HIV-1 IN drug resistance, which will be useful for designing anti-HIV inhibitors with unique resistance profiles. In this study, we use molecular dynamics simulations, within the hybrid quantum mechanics/molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy for wild-type and N155S mutant HIV-1 IN, both complexed with a DKA. This hybrid methodology has the advantage of the inclusion of quantum effects such as ligand polarization upon binding, which can be very important when highly polarizable groups are embedded in anisotropic environments, for example in metal-containing active sites. Furthermore, an energy terms decomposition analysis was performed to determine contributions of individual residues to the enzyme-inhibitor interactions. The results reveal that there is a strong interaction between the Lys-159, Lys-156, and Asn-155 residues and Mg(2+) cation and the DKA inhibitor. Our calculations show that the binding energy is higher in wild-type than in the N155S mutant, in accordance with the experimental results. The role of the mutated residue has thus been checked as maintaining the structure of the ternary complex formed by the protein, the Mg(2+) cation, and the inhibitor. These results might be useful to design compounds with more interesting anti-HIV-1 IN activity on the basis of its three-dimensional structure.
Assuntos
Aminobutiratos/química , Integrase de HIV/química , Integrase de HIV/ultraestrutura , Inibidores de Integrase/química , Modelos Químicos , Modelos Moleculares , Sítios de Ligação , Simulação por Computador , Integrase de HIV/genética , Magnésio/química , Mecânica , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Mutagênese Sítio-Dirigida , Fenilbutiratos , Ligação Proteica , Conformação Proteica , Teoria QuânticaRESUMO
Human immunodeficiency virus type-1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Diketo acids such as L-731,988 and S-1360 are potent and selective inhibitors of HIV-1 IN. In this study, we used molecular dynamics simulations, within the hybrid quantum mechanics/molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy between HIV-1 IN and L-731,988 and 10 of its derivatives and analogues. This hybrid methodology has the advantage that it includes quantum effects such as ligand polarisation upon binding, which can be very important when highly polarisable groups are embedded in anisotropic environments, as for example in metal-containing active sites. Furthermore, an energy decomposition analysis was performed to determine the contributions of individual residues to the enzyme-inhibitor interactions on averaged structures obtained from rather extensive conformational sampling. Analysis of the results reveals first that there is a correlation between protein-ligand interaction energy and experimental strand transfer into human chromosomes and secondly that the Asn-155, Lys-156 and Lys-159 residues and the Mg(2+) ion are crucial to anti-HIV IN activity. These results may explain the available experimental data.
Assuntos
Inibidores de Integrase de HIV/metabolismo , Integrase de HIV/metabolismo , Proteínas/metabolismo , Teoria Quântica , Inibidores de Integrase de HIV/química , Ligantes , Modelos MolecularesRESUMO
Integrase (IN) is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective viral replication. S-1360 is a potent and selective inhibitor of HIV-1 IN. In this work, we have carried out molecular dynamics (MD) simulations using a hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) approach, to determine the protein-ligand interaction energy for S-1360 and two analogues. Analysis of the MD trajectories reveals that the strongest protein-inhibitor interactions, observed in the three studied complexes, are established with Lys-159 residue and Mg(2+) cation. Calculations of binding energy using BLYP/MM level of theory reveal that there is a direct relationship between this theoretical computed property and the experimental determined anti-HIV activity.