RESUMO
The photochemically active sites of the proteins sfGFP66azF and Venus66azF, members of the green fluorescent protein (GFP) family, contain a non-canonical amino acid residue p-azidophenylalanine (azF) instead of Tyr66. The light-induced decomposition of azF at these sites leads to the formation of reactive arylnitrene (nF) intermediates followed by the formation of phenylamine-containing chromophores. We report the first study of the reaction mechanism of the reduction of the arylnitrene intermediates in sfGFP66nF and Venus66nF using molecular modeling methods. The Gibbs energy profiles for the elementary steps of the chemical reaction in sfGFP66nF are computed using molecular dynamics simulations with quantum mechanics/molecular mechanics (QM/MM) potentials. Structures and energies along the reaction pathway in Venus66nF are evaluated using a QM/MM approach. According to the results of the simulations, arylnitrene reduction is coupled with oxidation of the histidine side chain on the His148 residue located near the chromophore.
Assuntos
Azidas , Histidina , Proteínas de Fluorescência Verde/química , Histidina/química , Simulação de Dinâmica Molecular , Oxirredução , Corantes , Teoria QuânticaRESUMO
DNA aptamers are oligonucleotides that specifically bind to target molecules, similar to how antibodies bind to antigens. We identified an aptamer named MEZ that is highly specific to the receptor-binding domain, RBD, of the SARS-CoV-2 spike protein from the Wuhan-Hu-1 strain. The SELEX procedure was utilized to enrich the initial 31-mer oligonucleotide library with the target aptamer. The aptamer identification was performed using the novel protocol based on nanopore sequencing developed in this study. The MEZ aptamer was chemically synthesized and tested for binding with the SARS-CoV-2 RBD of the spike protein from different strains. The Kd is 6.5 nM for the complex with the RBD from the Wuhan-Hu-1 strain, which is comparable with known aptamers; the advantage is that the MEZ aptamer is smaller than known analogs. The proposed aptamer is highly selective for the RBD protein from the Wuhan-Hu-1 strain and does not form complexes with the RBD from Beta, Delta and Omicron strains. Experimental and theoretical studies together revealed the molecular mechanism of aptamer binding. The aptamer occupies the same binding site as ACE2 when bound to the RBD. The 3'-end of the MEZ aptamer is important for complex formation and is responsible for the discrimination of the RBD protein from a specific strain. The 5'-end is responsible for the formation of a loop in the 3D structure of the aptamer, which is important for proper binding.
Assuntos
Aptâmeros de Nucleotídeos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Aptâmeros de Nucleotídeos/química , SARS-CoV-2/química , SARS-CoV-2/isolamento & purificação , Humanos , Técnica de Seleção de Aptâmeros , Ligação Proteica , COVID-19/virologia , Sítios de LigaçãoRESUMO
The development of biocatalysts requires reorganization of the enzyme's active site to facilitate the productive binding of the target substrate and improve turnover number at desired conditions. Pyridoxal-5'-phosphate (PLP) - dependent transaminases are highly efficient biocatalysts for asymmetric amination of ketones and keto acids. However, transaminases, being stereoselective enzymes, have a narrow substrate specificity due to the ordered structure of the active site and work only in neutral-alkaline media. Here, we investigated the d-amino acid transaminase from Aminobacterium colombiense, with the active site organized differently from that of the canonical d-amino acid transaminase from Bacillus sp. YM-1. Using a combination of site-directed mutagenesis, kinetic analysis, molecular modeling, and structural analysis we determined the active site residues responsible for substrate binding, substrate differentiation, thermostability of a functional dimer, and affecting the pH optimum. We demonstrated that the high specificity toward d-glutamate/α-ketoglutarate is due to the interactions of a γ-carboxylate group with K237 residue, while binding of other substrates stems from the effectiveness of their accommodation in the active site optimized for d-glutamate/α-ketoglutarate binding. Furthermore, we showed that the K237A substitution shifts the catalytic activity optimum to acidic pH. Our findings are useful for achieving target substrate specificity and demonstrate the potential for developing and optimizing transaminases for various applications.
Assuntos
Aminoácidos , Transaminases , Transaminases/metabolismo , Ácidos Cetoglutáricos , Ácido Glutâmico , Especificidade por Substrato , Cinética , Concentração de Íons de HidrogênioRESUMO
The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k2/KS, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k2, and the weakening of the binding affinity, i.e., an increase in the substrate constant, KS. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2A501V variant exhibits a higher k2/KS value, whereas for PBP2A501R and PBP2A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k2/KS changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the ß-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k2/KS value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the ß3-ß4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k2/KS value. Thus, the experimentally observed decrease in the k2/KS in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k2, and the increase in KS.
Assuntos
Ceftriaxona , Simulação de Dinâmica Molecular , Neisseria gonorrhoeae , Proteínas de Ligação às Penicilinas , Ceftriaxona/farmacologia , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/metabolismo , Antibacterianos/farmacologia , Mutação , Farmacorresistência Bacteriana/genética , Acilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , D-Ala-D-Ala Carboxipeptidase Tipo SerinaRESUMO
Serine ß-lactamase TEM-1 is the first ß-lactamase discovered and is still common in Gram-negative pathogens resistant to ß-lactam antibiotics. It hydrolyzes penicillins and cephalosporins of early generations. Some of the emerging TEM-1 variants with one or several amino acid substitutions have even broader substrate specificity and resistance to known covalent inhibitors. Key amino acid substitutions affect catalytic properties of the enzyme, and secondary mutations accompany them. The occurrence of the secondary mutation M182T, called a "global suppressor", has almost doubled over the last decade. Therefore, we performed saturating mutagenesis at position 182 of TEM-1 to determine the influence of this single amino acid substitution on the catalytic properties, thermal stability, and ability for thermoreactivation. Steady-state parameters for penicillin, cephalothin, and ceftazidime are similar for all TEM-1 M182X variants, whereas melting temperature and ability to reactivate after incubation at a higher temperature vary significantly. The effects are multidirectional and depend on the particular amino acid at position 182. The M182E variant of ß-lactamase TEM-1 demonstrates the highest residual enzymatic activity, which is 1.5 times higher than for the wild-type enzyme. The 3D structure of the side chain of residue 182 is of particular importance as observed from the comparison of the M182I and M182L variants of TEM-1. Both of these amino acid residues have hydrophobic side chains of similar size, but their residual activity differs by three-fold. Molecular dynamic simulations add a mechanistic explanation for this phenomenon. The important structural element is the V159-R65-E177 triad that exists due to both electrostatic and hydrophobic interactions. Amino acid substitutions that disturb this triad lead to a decrease in the ability of the ß-lactamase to be reactivated.
Assuntos
Substituição de Aminoácidos , Estabilidade Enzimática , beta-Lactamases , beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismo , Metionina/química , Metionina/metabolismo , Metionina/genética , Modelos Moleculares , Mutagênese , Cinética , Simulação de Dinâmica Molecular , Penicilinas/química , Penicilinas/metabolismoRESUMO
We report the results of computational modeling of the reactions of the SARS-CoV-2 main protease (MPro) with four potential covalent inhibitors. Two of them, carmofur and nirmatrelvir, have shown experimentally the ability to inhibit MPro. Two other compounds, X77A and X77C, were designed computationally in this work. They were derived from the structure of X77, a non-covalent inhibitor forming a tight surface complex with MPro. We modified the X77 structure by introducing warheads capable of reacting with the catalytic cysteine residue in the MPro active site. The reaction mechanisms of the four molecules with MPro were investigated by quantum mechanics/molecular mechanics (QM/MM) simulations. The results show that all four compounds form covalent adducts with the catalytic cysteine Cys 145 of MPro. From the chemical perspective, the reactions of these four molecules with MPro follow three distinct mechanisms. The reactions are initiated by a nucleophilic attack of the thiolate group of the deprotonated cysteine residue from the catalytic dyad Cys145-His41 of MPro. In the case of carmofur and X77A, the covalent binding of the thiolate to the ligand is accompanied by the formation of the fluoro-uracil leaving group. The reaction with X77C follows the nucleophilic aromatic substitution SNAr mechanism. The reaction of MPro with nirmatrelvir (which has a reactive nitrile group) leads to the formation of a covalent thioimidate adduct with the thiolate of the Cys145 residue in the enzyme active site. Our results contribute to the ongoing search for efficient inhibitors of the SARS-CoV-2 enzymes.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Cisteína , Simulação de Dinâmica Molecular , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Antivirais/farmacologia , Simulação de Acoplamento MolecularRESUMO
Biocatalytic copper centers are generally involved in the activation and reduction of dioxygen, with only few exceptions known. Here we report the discovery and characterization of a previously undescribed copper center that forms the active site of a copper-containing enzyme thiocyanate dehydrogenase (suggested EC 1.8.2.7) that was purified from the haloalkaliphilic sulfur-oxidizing bacterium of the genus Thioalkalivibrio ubiquitous in saline alkaline soda lakes. The copper cluster is formed by three copper ions located at the corners of a near-isosceles triangle and facilitates a direct thiocyanate conversion into cyanate, elemental sulfur, and two reducing equivalents without involvement of molecular oxygen. A molecular mechanism of catalysis is suggested based on high-resolution three-dimensional structures, electron paramagnetic resonance (EPR) spectroscopy, quantum mechanics/molecular mechanics (QM/MM) simulations, kinetic studies, and the results of site-directed mutagenesis.
Assuntos
Proteínas de Bactérias/química , Domínio Catalítico , Cobre/química , Ectothiorhodospiraceae/enzimologia , Oxirredutases/química , Bactérias Redutoras de Enxofre/enzimologia , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Oxirredução , Oxigênio/química , Enxofre/químicaRESUMO
The mRubyFT is a monomeric genetically encoded fluorescent timer based on the mRuby2 fluorescent protein, which is characterized by the complete maturation of the blue form with the subsequent conversion to the red one. It has higher brightness in mammalian cells and higher photostability compared with other fluorescent timers. A high-resolution structure is a known characteristic of the mRubyFT with the red form chromophore, but structural details of its blue form remain obscure. In order to obtain insight into this, we obtained an S148I variant of the mRubyFT (mRubyFTS148I) with the blocked over time blue form of the chromophore. X-ray data at a 1.8 Å resolution allowed us to propose a chromophore conformation and its interactions with the neighboring residues. The imidazolidinone moiety of the chromophore is completely matured, being a conjugated π-system. The methine bridge is not oxidized in the blue form bringing flexibility to the phenolic moiety that manifests itself in poor electron density. Integration of these data with the results of molecular dynamic simulation disclosed that the OH group of the phenolic moiety forms a hydrogen bond with the side chain of the T163 residue. A detailed comparison of mRubyFTS148I with other available structures of the blue form of fluorescent proteins, Blue102 and mTagBFP, revealed a number of characteristic differences. Molecular dynamic simulations with the combined quantum mechanic/molecular mechanic potentials demonstrated that the blue form exists in two protonation states, anion and zwitterion, both sharing enolate tautomeric forms of the C=C-O- fragment. These two forms have similar excitation energies, as evaluated by calculations. Finally, excited state molecular dynamic simulations showed that excitation of the chromophore in both protonation states leads to the same anionic fluorescent state. The data obtained shed light on the structural features and spectral properties of the blue form of the mRubyFT timer.
Assuntos
Corantes , Simulação de Dinâmica Molecular , Proteínas Luminescentes/metabolismo , Proteínas de Fluorescência Verde/químicaRESUMO
Integration of HIV-1 genomic cDNA results in the formation of single-strand breaks in cellular DNA, which must be repaired for efficient viral replication. Post-integration DNA repair mainly depends on the formation of the HIV-1 integrase complex with the Ku70 protein, which promotes DNA-PK assembly at sites of integration and its activation. Here, we have developed a first-class inhibitor of the integrase-Ku70 complex formation that inhibits HIV-1 replication in cell culture by acting at the stage of post-integration DNA repair. This inhibitor, named s17, does not affect the main cellular function of Ku70, namely its participation in the repair of double-strand DNA breaks through the non-homologous end-joining pathway. Using a molecular dynamics approach, we have constructed a model for the interaction of s17 with Ku70. According to this model, the interaction of two phenyl radicals of s17 with the L76 residue of Ku70 is important for this interaction. The requirement of two phenyl radicals in the structure of s17 for its inhibitory properties was confirmed using a set of s17 derivatives. We propose to stimulate compounds that inhibit post-integration repair by disrupting the integrase binding to Ku70 KuINins.
Assuntos
HIV-1 , HIV-1/fisiologia , Autoantígeno Ku/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA , Integrases/metabolismo , Reparo do DNA por Junção de ExtremidadesRESUMO
Pyridoxal-5'-phosphate (PLP)-dependent transaminases are highly efficient biocatalysts for stereoselective amination. D-amino acid transaminases can catalyze stereoselective transamination producing optically pure D-amino acids. The knowledge of substrate binding mode and substrate differentiation mechanism in D-amino acid transaminases comes down to the analysis of the transaminase from Bacillus subtilis. However, at least two groups of D-amino acid transaminases differing in the active site organization are known today. Here, we present a detailed study of D-amino acid transaminase from the gram-negative bacterium Aminobacterium colombiense with a substrate binding mode different from that for the transaminase from B. subtilis. We study the enzyme using kinetic analysis, molecular modeling, and structural analysis of holoenzyme and its complex with D-glutamate. We compare the multipoint binding of D-glutamate with the binding of other substrates, D-aspartate and D-ornithine. QM/MM MD simulation reveals that the substrate can act as a base and its proton can be transferred from the amino group to the α-carboxylate group. This process occurs simultaneously with the nucleophilic attack of the PLP carbon atom by the nitrogen atom of the substrate forming gem-diamine at the transimination step. This explains the absence of the catalytic activity toward (R)-amines that lack an α-carboxylate group. The obtained results clarify another substrate binding mode in D-amino acid transaminases and underpinned the substrate activation mechanism.
Assuntos
Aminoácidos , Transaminases , Transaminases/metabolismo , Ácido Glutâmico , Cinética , Bacillus subtilis/metabolismo , Fosfato de Piridoxal/metabolismo , Catálise , Especificidade por SubstratoRESUMO
The equilibrium between keto and enol forms in acetylacetone and its derivatives is studied using electron delocalization indices and delocalization tensor density. We demonstrate how electron delocalization governs the equilibrium between keto and enol forms. The less stable enols have more distinct double and single bond character in the CCC fragment, while electron delocalization in this fragment is more pronounced in more stable enols. Looking for the origin of such behavior, we considered the one-electron potentials entering the Euler equation for the electron density. We found that electron delocalization is mainly governed by the static exchange potential, which depends on the three-dimensional atomic structure. It, however, does not distinguish differences in electron delocalization in more and less stable enols, the effect arising from the kinetic exchange contribution, which reflects spin-dependent effects in the electron motion. The local depletion of kinetic exchange in the conjugated fragment yields the enhanced electron delocalization along the CCC bonds in more stable enols. Thus, a combination of considered descriptors allowed us to reveal the influence of electron delocalization on the equilibrium between keto and enol forms and showed the significant features of this phenomenon.
Assuntos
Álcoois , Elétrons , CinéticaRESUMO
Deactivation of the ß-lactam antibiotics in the active sites of the ß-lactamases is among the main mechanisms of bacterial antibiotic resistance. As drugs of last resort, carbapenems are efficiently hydrolyzed by metallo-ß-lactamases, presenting a serious threat to human health. Our study reveals mechanistic aspects of the imipenem hydrolysis by bizinc metallo-ß-lactamases, NDM-1 and L1, belonging to the B1 and the B3 subclasses, respectively. The results of QM(PBE0-D3/6-31G**)/MM simulations show that the enamine product with the protonated nitrogen atom is formed as the major product in NDM-1 and as the only product in the L1 active site. In NDM-1, there is also another reaction pathway that leads to the formation of the (S)-enantiomer of the imine form of the hydrolyzed imipenem; this process occurs with the higher energy barriers. The absence of the second pathway in L1 is due to the different amino acid composition of the active site loop. In L1, the hydrophobic Pro226 residue is located above the pyrroline ring of imipenem that blocks protonation of the carbon atom. Electron density analysis is performed at the stationary points to compare reaction pathways in L1 and NDM-1. Tautomerization from the enamine to the imine form likely happens in solution after the dissociation of the hydrolyzed imipenem from the active site of the enzyme. Classical molecular dynamics simulations of the hydrolyzed imipenem in solution, both with the neutral enamine and the negatively charged N-C2-C3 fragment, demonstrate a huge diversity of conformations. The vast majority of conformations blocks the C3-atom from the side required for the (S)-imine formation upon tautomerization. Thus, according to our calculations, formation of the (R)-imine is more likely. QM(PBE0-D3/6-31G**)/MM molecular dynamics simulations of the hydrolyzed imipenem with the negatively charged N-C2-C3 fragment followed by the Laplacian bond order analysis demonstrate that the NâC2-C3- resonance structure is the most pronounced that facilitates formation of the imine form. The proposed mechanism of the enzymatic enamine formation and its subsequent tautomerization to the imine form in solution is in agreement with the recent spectroscopic and NMR studies.
Assuntos
Imipenem , beta-Lactamases , Humanos , Imipenem/química , Imipenem/metabolismo , beta-Lactamases/química , Domínio Catalítico , Iminas/química , Simulação de Dinâmica Molecular , Água , Antibacterianos/químicaRESUMO
Penicillin-binding proteins 2 (PBP2) are critically important enzymes in the formation of the bacterial cell wall. Inhibition of PBP2 is utilized in the treatment of various diseases, including gonorrhea. Ceftriaxone is the only drug used to treat gonorrhea currently, and recent growth in PBP2 resistance to this antibiotic is a serious threat to human health. Our study reveals mechanistic aspects of the inhibition reaction of PBP2 from the wild-type FA19 strain and mutant 35/02 and H041 strains of Neisseria Gonorrhoeae by ceftriaxone. QM(PBE0-D3/6-31G**)/MM MD simulations show that the reaction mechanism for the wild-type PBP2 consists of three elementary steps including nucleophilic attack, C-N bond cleavage in the ß-lactam ring and elimination of the leaving group in ceftriaxone. In PBP2 from the mutant strains, the second and third steps occur simultaneously. For all considered systems, the acylation rate is determined by the energy barrier of the first step that increases in the order of PBP2 from FA19, 35/02 and H041 strains. Dynamic behavior of ES complexes is analyzed using geometry and electron density features including Fukui electrophilicity index and Laplacian of electron density maps. It reveals that more efficient activation of the carbonyl group of the antibiotic leads to the lower energy barrier of nucleophilic attack and larger stabilization of the first reaction intermediate. Dynamical network analysis of MD trajectories explains the differences in ceftriaxone binding affinity: in PBP2 from the wild-type strain, the ß3-ß4 loop conformation facilitates substrate binding, whereas in PBP2 from the mutant strains, it exists in the conformation that is unfavorable for complex formation. Thus, we clarify that the experimentally observed decrease in the second-order rate constant of acylation (k2/KS) in PBP2 from the mutant strains is due to both a decrease in the acylation rate constant k2 and an increase in the dissociation constant KS.
Assuntos
Ceftriaxona , Gonorreia , Humanos , Ceftriaxona/farmacologia , Proteínas de Ligação às Penicilinas/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Neisseria gonorrhoeae/genética , Testes de Sensibilidade Microbiana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
Nanopore sequencing (ONT) is a new and rapidly developing method for determining nucleotide sequences in DNA and RNA. It serves the ability to obtain long reads of thousands of nucleotides without assembly and amplification during sequencing compared to next-generation sequencing. Nanopore sequencing can help for determination of genetic changes leading to antibiotics resistance. This study presents the application of ONT technology in the assembly of an E. coli genome characterized by a deletion of the tolC gene and known single-nucleotide variations leading to antibiotic resistance, in the absence of a reference genome. We performed benchmark studies to determine minimum coverage depth to obtain a complete genome, depending on the quality of the ONT data. A comparison of existing programs was carried out. It was shown that the Flye program demonstrates plausible assembly results relative to others (Shasta, Canu, and Necat). The required coverage depth for successful assembly strongly depends on the size of reads. When using high-quality samples with an average read length of 8 Kbp or more, the coverage depth of 30× is sufficient to assemble the complete genome de novo and reliably determine single-nucleotide variations in it. For samples with shorter reads with mean lengths of 2 Kbp, a higher coverage depth of 50× is required. Avoiding of mechanical mixing is obligatory for samples preparation. Nanopore sequencing can be used alone to determine antibiotics-resistant genetic features of bacterial strains.
Assuntos
Sequenciamento por Nanoporos , Antibacterianos/farmacologia , Escherichia coli/genética , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodosRESUMO
The increasing antibiotic resistance is a clinical problem worldwide. Numerous Gram-negative bacteria have already become resistant to the most widely used class of antibacterial drugs, ß-lactams. One of the main mechanisms is inactivation of ß-lactam antibiotics by bacterial ß-lactamases. Appearance and spread of these enzymes represent a continuous challenge for the clinical treatment of infections and for the design of new antibiotics and inhibitors. Drug repurposing is a prospective approach for finding new targets for drugs already approved for use. We describe here the inhibitory potency of known detoxifying antidote 2,3-dimercaptopropane-1-sulfonate (unithiol) against metallo-ß-lactamases. Unithiol acts as a competitive inhibitor of meropenem hydrolysis by recombinant metallo-ß-lactamase NDM-1 with the KI of 16.7 µM. It is an order of magnitude lower than the KI for l-captopril, the inhibitor of angiotensin-converting enzyme approved as a drug for the treatment of hypertension. Phenotypic methods demonstrate that the unithiol inhibits natural metallo-ß-lactamases NDM-1 and VIM-2 produced by carbapenem-resistant K. pneumoniae and P. aeruginosa bacterial strains. The 3D full atom structures of unithiol complexes with NDM-1 and VIM-2 are obtained using QM/MM modeling. The thiol group is located between zinc cations of the active site occupying the same place as the catalytic hydroxide anion in the enzyme-substrate complex. The sulfate group forms both a coordination bond with a zinc cation and hydrogen bonds with the positively charged residue, lysine or arginine, responsible for proper orientation of antibiotics upon binding to the active site prior to hydrolysis. Thus, we demonstrate both experimentally and theoretically that the unithiol is a prospective competitive inhibitor of metallo-ß-lactamases and it can be utilized in complex therapy together with the known ß-lactam antibiotics.
Assuntos
Klebsiella pneumoniae/enzimologia , Pseudomonas aeruginosa/enzimologia , Unitiol/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Carbapenêmicos/farmacologia , Reposicionamento de Medicamentos , Farmacorresistência Bacteriana/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Modelos Moleculares , Conformação Proteica , Pseudomonas aeruginosa/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , beta-Lactamases/químicaRESUMO
The influence of the active site flexibility on the efficiency of catalytic reaction is studied by taking two members of metallo-ß-lactamases, L1 and NDM-1, with the same substrate, imipenem. Active sites of these proteins are covered by L10 loops, and differences in their amino acid compositions affect their rigidity. A more flexible loop in the NDM-1 brings additional flexibility to the active site in the ES complex. This is pronounced in wider distributions of key interatomic distances, such as the distance of the nucleophilic attack, coordination bond lengths, and covalent bond lengths in the substrate. Substrate activation, quantified by Fukui electrophilicity index of the carbonyl carbon atom of the substrate, is also sensitive to the active site flexibility. In the tighter and more rigid L1 enzyme-substrate complex, the substrate is activated more efficiently. In the NDM-1 containing system, only one third of the states are activated to the same extent. Other fractions demonstrate lower substrate activation. Efficiency of the substrate activation and rigidity of the ES complex influence the following chemical reaction. In the more rigid L1-containing system, the reaction barrier of the first step of the reaction is lower, and the first intermediate is more stabilized compared to the NDM-1 containing system.
Assuntos
Zinco , beta-Lactamases , beta-Lactamases/química , Domínio Catalítico , Zinco/química , Imipenem , Aminoácidos , CarbonoRESUMO
We applied a set of advanced bonding descriptors to establish the hidden electron density features and binding energy characteristics of intermolecular DHâââA hydrogen bonds (OHâââO, NHâââO and SHâââO) in 150 isolated and solvated molecular complexes. The exchange-correlation and Pauli potentials as well as corresponding local one-electron forces allowed us to explicitly ascertain how electron exchange defines the bonding picture in the proximity of the H-bond critical point. The electron density features of DHâââA interaction are governed by alterations in the electron localization in the H-bond region displaying itself in the exchange hole. At that, they do not depend on the variations in the exchange hole mobility. The electrostatic interaction mainly defines the energy of H-bonds of different types, whereas the strengthening/weakening of H-bonds in complexes with varying substituents depends on the barrier height of the exchange potential near the bond critical point. Energy variations between H-bonds in isolated and solvated systems are also caused the electron exchange peculiarities as follows from the corresponding potential and the interacting quantum atom analyses complemented by electron delocalization index calculations. Our approach is based on the bonding descriptors associated with the characteristics of the observable electron density and can be recommended for in-depth studies of non-covalent bonding.
RESUMO
We describe a model for spectral tuning in red fluorescent proteins (RFPs) based on the relation between an electronic structure descriptor, the dipole moment variation upon excitation (DMV), and the excitation energy of a protein. This approach aims to overcome the problem of accurate prediction of excitation energies in RFPs, which span a very narrow window of band maxima. The latter roughly corresponds to the energy range of 0.1 eV, which is comparable with typical errors in calculations of the excitation energy by conventional quantum chemistry methods. In this work, we demonstrate a strong quantitative correlation between DMV values, obtained computationally with modest efforts, and excitation energies ΔEex at the experimental excitation band maxima for a series of RFPs with bands between 570 and 605 nm. Protein models are constructed by motifs of the relevant crystal structures, and atomic coordinates are optimized in quantum mechanics/molecular mechanics (QM/MM) calculations with QM-subsystems composed of large chromophore-containing regions. DMV values are evaluated with the electron density computed at the time-dependent density functional theory (TDDFT) level using several functionals and basis sets. We show that the results obtained with the CAM-B3LYP, BHHLYP, and M06-2X functionals demonstrate favorable correlations between DMV and ΔEex with the mean absolute error less than 0.01 eV. Taking into account the solid theoretical grounds of the relation between the DMV and the excitation energy in fluorescent proteins, the described modeling strategy presents a rational tool for spectral tuning in these efficient markers for in vivo imaging.
Assuntos
Simulação de Dinâmica Molecular , Teoria Quântica , Teoria da Densidade FuncionalRESUMO
We report the first computational characterization of an optogenetic system composed of two photosensing BLUF (blue light sensor using flavin adenine dinucleotide) domains and two catalytic adenylyl cyclase (AC) domains. Conversion of adenosine triphosphate (ATP) to the reaction products, cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi), catalyzed by ACs initiated by excitation in photosensing domains has emerged in the focus of modern optogenetic applications because of the request in photoregulated enzymes that modulate cellular concentrations of signaling messengers. The photoactivated AC from the soil bacterium Beggiatoa sp. (bPAC) is an important model showing a considerable increase in the ATP to cAMP conversion rate in the catalytic domain after the illumination of the BLUF domain. The 1 µs classical molecular dynamics simulations reveal that the activation of the BLUF domain leading to tautomerization of Gln49 in the chromophore-binding pocket results in switching of the position of the side chain of Arg278 in the active site of AC. Allosteric signal transmission pathways between Gln49 from BLUF and Arg278 from AC were revealed by the dynamical network analysis. The Gibbs energy profiles of the ATP â cAMP + PPi reaction computed using QM(DFT(ωB97X-D3/6-31G**))/MM(CHARMM) molecular dynamics simulations for both Arg278 conformations in AC clarify the reaction mechanism. In the light-activated system, the corresponding arginine conformation stabilizes the pentacoordinated phosphorus of the α-phosphate group in the transition state, thus lowering the activation energy. Simulations of the bPAC system with the Tyr7Phe replacement in the BLUF demonstrate occurrence of both arginine conformations in an equal ratio, explaining the experimentally observed intermediate catalytic activity of the bPAC-Y7F variant as compared with the dark and light states of the wild-type bPAC.
Assuntos
Adenilil Ciclases , Optogenética , Monofosfato de Adenosina , Trifosfato de Adenosina , Adenilil Ciclases/genética , Arginina , Proteínas de Bactérias/genética , LuzRESUMO
The review focuses on bacterial metallo-ß-lactamases (MßLs) responsible for the inactivation of ß-lactams and associated antibiotic resistance. The diversity of the active site structure in the members of different MßL subclasses explains different mechanisms of antibiotic hydrolysis and should be taken into account when searching for potential MßL inhibitors. The review describes the features of the antibiotic inactivation mechanisms by various MßLs studied by X-ray crystallography, NMR, kinetic measurements, and molecular modeling. The mechanisms of enzyme inhibition for each MßL subclass are discussed.