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Accounts of Chemical Research Jan 2022Non-heme iron dioxygenases catalyze vital processes for human health related to the biosynthesis of essential products and the biodegradation of toxic metabolites. Often... (Review)
Review
Local Charge Distributions, Electric Dipole Moments, and Local Electric Fields Influence Reactivity Patterns and Guide Regioselectivities in α-Ketoglutarate-Dependent Non-heme Iron Dioxygenases.
Non-heme iron dioxygenases catalyze vital processes for human health related to the biosynthesis of essential products and the biodegradation of toxic metabolites. Often the natural product biosyntheses by these non-heme iron dioxygenases is highly regio- and chemoselective, which are commonly assigned to tight substrate-binding and positioning. However, recent high-level computational modeling has shown that substrate-binding and positioning is only part of the story and long-range electrostatic interactions can play a major additional role.In this Account, we review and summarize computational viewpoints on the high regio- and chemoselectivity of α-ketoglutarate-dependent non-heme iron dioxygenases and how external perturbations affect the catalysis. In particular, studies from our groups have shown that often a regioselectivity in enzymes can be accomplished by stabilization of the rate-determining transition state for the reaction through external charges, electric dipole moments, or local electric field effects. Furthermore, bond dissociation energies in molecules are shown to be influenced by an electric field effect, and through targeting a specific bond in an electric field, this can lead to an unusually specific reaction. For instance, in the carbon-induced starvation protein, we studied two substrate-bound conformations and showed that regardless of what C-H bond of the substrate is closest to the iron(IV)-oxo oxidant, the lowest hydrogen atom abstraction barrier is always for the pro- C-H abstraction due to an induced dipole moment of the protein that weakens this bond. In another example of the hygromycin biosynthesis enzyme, an oxidative ring-closure reaction in the substrate forms an ortho-δ-ester ring. Calculations on this enzyme show that the selectivity is guided by a protonated lysine residue in the active site that, through its positive charge, triggers a low energy hydrogen atom abstraction barrier. A final set of examples in this Account discuss the viomycin biosynthesis enzyme and the 2-(trimethylammonio)ethylphosphonate dioxygenase (TmpA) enzyme. Both of these enzymes are shown to possess a significant local dipole moment and local electric field effect due to charged residues surrounding the substrate and oxidant binding pockets. The protein dipole moment and local electric field strength changes the C-H bond strengths of the substrate as compared to the gas-phase triggers the regioselectivity of substrate activation. In particular, we show that in the gas phase and in a protein environment C-H bond strengths are different due to local electric dipole moments and electric field strengths. These examples show that enzymes have an intricately designed structure that enables a chemical reaction under ambient conditions through the positioning of positively and negatively charged residues that influence and enhance reaction mechanisms. These computational insights create huge possibilities in bioengineering to apply local electric field and dipole moments in proteins to achieve an unusual selectivity and specificity and trigger a fit-for-purpose biocatalyst for unique biotransformations.
Topics: Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Catalytic Domain; Dioxygenases; Humans; Iron; Ketoglutaric Acids
PubMed: 34915695
DOI: 10.1021/acs.accounts.1c00538 -
International Journal of Molecular... Sep 2021The growth of the polypeptide chain occurs due to the fast and coordinated work of the ribosome and protein elongation factors, EF-Tu and EF-G. However, the exact...
The growth of the polypeptide chain occurs due to the fast and coordinated work of the ribosome and protein elongation factors, EF-Tu and EF-G. However, the exact contribution of each of these components in the overall balance of translation kinetics remains not fully understood. We created an in vitro translation system replacing either elongation factor with heterologous thermophilic protein from . The rates of the A-site binding and decoding reactions decreased an order of magnitude in the presence of thermophilic EF-Tu, indicating that the kinetics of aminoacyl-tRNA delivery depends on the properties of the elongation factor. On the contrary, thermophilic EF-G demonstrated the same translocation kinetics as a mesophilic protein. Effects of translocation inhibitors (spectinomycin, hygromycin B, viomycin and streptomycin) were also similar for both proteins. Thus, the process of translocation largely relies on the interaction of tRNAs and the ribosome and can be efficiently catalysed by thermophilic EF-G even at suboptimal temperatures.
Topics: Bacterial Proteins; Escherichia coli; Peptide Chain Elongation, Translational; Peptide Elongation Factor G; Peptide Elongation Factor Tu; RNA, Bacterial; RNA, Transfer; Ribosomes; Thermus thermophilus
PubMed: 34502523
DOI: 10.3390/ijms22179614 -
Journal of Biomolecular Structure &... 2022The current outbreak of COVID-19 is leading an unprecedented scientific effort focusing on targeting SARS-CoV-2 proteins critical for its viral replication. Herein, we...
Quantitative structure-activity relationships, molecular docking and molecular dynamics simulations reveal drug repurposing candidates as potent SARS-CoV-2 main protease inhibitors.
The current outbreak of COVID-19 is leading an unprecedented scientific effort focusing on targeting SARS-CoV-2 proteins critical for its viral replication. Herein, we performed high-throughput virtual screening of more than eleven thousand FDA-approved drugs using backpropagation-based artificial neural networks ( = 0.60, = 0.80 and = 0.91), partial-least-square (PLS) regression ( = 0.83, = 0.62 and = 0.70) and sequential minimal optimization (SMO) regression ( = 0.70, = 0.80 and = 0.89). We simulated the stability of Acarbose-derived hexasaccharide, Naratriptan, Peramivir, Dihydrostreptomycin, Enviomycin, Rolitetracycline, Viomycin, Angiotensin II, Angiotensin 1-7, Angiotensinamide, Fenoterol, Zanamivir, Laninamivir and Laninamivir octanoate with 3CL by 100 ns and calculated binding free energy using molecular mechanics combined with Poisson-Boltzmann surface area (MM-PBSA). Our QSAR models and molecular dynamics data suggest that seven repurposed-drug candidates such as Acarbose-derived Hexasaccharide, Angiotensinamide, Dihydrostreptomycin, Enviomycin, Fenoterol, Naratriptan and Viomycin are potential SARS-CoV-2 main protease inhibitors. In addition, our QSAR models and molecular dynamics simulations revealed that His41, Asn142, Cys145, Glu166 and Gln189 are potential pharmacophoric centers for 3CL inhibitors. Glu166 is a potential pharmacophore for drug design and inhibitors that interact with this residue may be critical to avoid dimerization of 3CL. Our results will contribute to future investigations of novel chemical scaffolds and the discovery of novel hits in high-throughput screening as potential anti-SARS-CoV-2 properties.Communicated by Ramaswamy H. Sarma.
Topics: Acarbose; Angiotensin Amide; Dihydrostreptomycin Sulfate; Drug Repositioning; Enviomycin; Fenoterol; Molecular Docking Simulation; Molecular Dynamics Simulation; Protease Inhibitors; Quantitative Structure-Activity Relationship; SARS-CoV-2; Antiviral Agents
PubMed: 34370631
DOI: 10.1080/07391102.2021.1958700 -
RNA (New York, N.Y.) Sep 2021Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the...
Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA movement), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, their kinetic effect on the EF-G-induced transition to the rotated/swiveled state of the SSU is moderate. The antibiotics mostly inhibit backward movements of the SSU body and/or the head domains. Vio, Spc, and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB, and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action.
Topics: Anti-Bacterial Agents; Biological Transport; Cinnamates; Escherichia coli; Hygromycin B; Kanamycin; Kinetics; Neomycin; Paromomycin; Peptide Elongation Factor G; Protein Biosynthesis; RNA, Messenger; RNA, Transfer; Ribosome Subunits; Spectinomycin; Streptomycin; Viomycin
PubMed: 34117118
DOI: 10.1261/rna.078758.121 -
The Journal of Physical Chemistry. A Mar 2021The viomycin biosynthesis enzyme VioC is a nonheme iron and α-ketoglutarate-dependent dioxygenase involved in the selective hydroxylation of l-arginine at the...
How Do Electrostatic Perturbations of the Protein Affect the Bifurcation Pathways of Substrate Hydroxylation versus Desaturation in the Nonheme Iron-Dependent Viomycin Biosynthesis Enzyme?
The viomycin biosynthesis enzyme VioC is a nonheme iron and α-ketoglutarate-dependent dioxygenase involved in the selective hydroxylation of l-arginine at the C-position for antibiotics biosynthesis. Interestingly, experimental studies showed that using the substrate analogue, namely, l-homo-arginine, a mixture of products was obtained originating from C-hydroxylation, C-hydroxylation, and C-C-desaturation. To understand how the addition of one CH group to a substrate can lead to such a dramatic change in selectivity and activity, we decided to perform a computational study using quantum mechanical (QM) cluster models. We set up a large active-site cluster model of 245 atoms that includes the oxidant with its first- and second-coordination sphere influences as well as the substrate binding pocket. The model was validated against experimental work from the literature on related enzymes and previous computational studies. Thereafter, possible pathways leading to products and byproducts were investigated for a model containing l-Arg and one for l-homo-Arg as substrate. The calculated free energies of activation predict product distributions that match the experimental observation and give a low-energy C-hydroxylation pathway for l-Arg, while for l-homo-Arg, several barriers are found to be close in energy leading to a mixture of products. We then analyzed the origins of the differences in product distributions using thermochemical, valence bond, and electrostatic models. Our studies show that the C-H and C-H bond strengths of l-Arg and l-homo-Arg are similar; however, external perturbations from an induced electric field of the protein affect the relative C-H bond strengths of l-Arg dramatically and make the C-H bond the weakest and guide the reaction to a selective C-hydroxylation channel. Therefore, the charge distribution in the protein and the induced electric dipole field of the active site of VioC guides the l-Arg substrate activation to C-hydroxylation and disfavors the C-hydroxylation pathway, while this does not occur for l-homo-Arg. Tight substrate positioning and electrostatic perturbations from the second-coordination sphere residues in VioC also result in a slower overall reaction for l-Arg; however, they enable a high substrate selectivity. Our studies highlight the importance of the second-coordination sphere in proteins that position the substrate and oxidant, perturb charge distributions, and enable substrate selectivity.
Topics: Bacterial Proteins; Catalytic Domain; Hydroxylation; Iron; Models, Molecular; Nonheme Iron Proteins; Oxygenases; Static Electricity; Viomycin
PubMed: 33620220
DOI: 10.1021/acs.jpca.1c00141 -
Biochemistry Jan 2021Capreomycin (CMN) and viomycin (VIO) are nonribosomal peptide antituberculosis antibiotics, the structures of which contain four nonproteinogenic amino acids, including...
Capreomycin (CMN) and viomycin (VIO) are nonribosomal peptide antituberculosis antibiotics, the structures of which contain four nonproteinogenic amino acids, including l-2,3-diaminopropionic acid (l-Dap), β-ureidodehydroalanine, l-capreomycidine, and β-lysine. Previous bioinformatics analysis suggested that CmnB/VioB and CmnK/VioK participate in the formation of l-Dap; however, the real substrates of these enzymes are yet to be confirmed. We herein show that starting from -phospho-l-Ser (OPS) and l-Glu precursors, CmnB catalyzes the condensation reaction to generate a metabolite intermediate -(1-amino-1-carboxyl-2-ethyl)glutamic acid (ACEGA), which undergoes NAD-dependent oxidative hydrolysis by CmnK to generate l-Dap. Furthermore, the binding site of ACEGA and the catalytic mechanism of CmnK were elucidated with the assistance of three crystal structures, including those of apo-CmnK, the NAD-CmnK complex, and CmnK in an alternative conformation. The CmnK-ACEGA docking model revealed that the glutamate α-hydrogen points toward the nicotinamide moiety. It provides evidence that the reaction is dependent on hydride transfer to form an imine intermediate, which is subsequently hydrolyzed by a water molecule to produce l-Dap. These findings modify the original proposed pathway and provide insights into l-Dap formation in the biosynthesis of other related natural products.
Topics: Aminobutyrates; Bacterial Proteins; Binding Sites; Capreomycin; Catalysis; Crystallography, X-Ray; Hydrolysis; Models, Molecular; Streptomyces; Substrate Specificity
PubMed: 33356147
DOI: 10.1021/acs.biochem.0c00808 -
Bioresource Technology Dec 20203-Hydroxyarginine (3-OH-Arg) is an important intermediate for the synthesis of viomycin, an important antibiotic for the clinical treatment of tuberculosis. An efficient...
3-Hydroxyarginine (3-OH-Arg) is an important intermediate for the synthesis of viomycin, an important antibiotic for the clinical treatment of tuberculosis. An efficient strategy for 3-OH-Arg production based on protein engineering and recombinant whole-cell biocatalysis was demonstrated for the first time. To avoid challenging product separation due to the generation of α-ketoglutarate (α-KG) in the system, the molar ratio of the substrates L-Arg and L-Glu was optimized to ensure the efficient production of 3-OH-Arg as well as the complete consumption of α-KG. Through the establishment of a fed-batch process, 3-OH-Arg and succinic acid (SA) production reached to 9.9 g/L and 5.98 g/L after 36 h of reaction under the optimized conditions. This is the highest biosynthetic yield of 3-OH-Arg achieved to date, potentially offering a promising strategy for commercial production of hydroxylated amino acids.
Topics: Biocatalysis; Ketoglutaric Acids; Protein Engineering; Succinic Acid
PubMed: 33099094
DOI: 10.1016/j.biortech.2020.124261 -
Journal of Biomolecular Structure &... Feb 20223CL is the main protease of the novel coronavirus (SARS-CoV-2) responsible for their intracellular duplication. Based on virtual screening technology and molecular...
3CL is the main protease of the novel coronavirus (SARS-CoV-2) responsible for their intracellular duplication. Based on virtual screening technology and molecular dynamics simulation, we found 23 approved clinical drugs such as Viomycin, Capastat, Carfilzomib and Saquinavir, which showed high affinity with the 3CL active sites. These findings showed that there were potential drugs that inhibit SARS-Cov-2's 3CL in the current clinical drug library, and these drugs can be further tested or chemically modified for the treatment of COVID-19.Communicated by Ramaswamy H. Sarma.
Topics: COVID-19; Humans; Molecular Docking Simulation; Peptide Hydrolases; Pharmaceutical Preparations; Protease Inhibitors; SARS-CoV-2
PubMed: 32909528
DOI: 10.1080/07391102.2020.1817786 -
Journal of Biomolecular Structure &... Jul 2021The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which was first reported in Wuhan province of China, has become a deadly pandemic causing alarmingly...
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which was first reported in Wuhan province of China, has become a deadly pandemic causing alarmingly high morbidity and mortality. In the absence of new targeted drugs and vaccines against SARS-CoV-2 at present, the choices for effective treatments are limited. Therefore, considering the exigency of the situation, we focused on identifying the available approved drugs as potential inhibitor against the promising Coronavirus drug target, the Main Protease, using computer-aided methods. We created a library of U. S. Food and Drug Administration approved anti-microbial drugs and virtually screened it against the available crystal structures of Main Protease of the virus. The study revealed that Viomycin showed the highest -CDocker energy after docking at the active site of SARS-CoV-2 Main Protease. It is noteworthy that Viomycin showed higher -CDocker energy as compared to the drugs currently under clinical trial for SARS-CoV-2 treatment Ritonavir and Lopinavir. Additionally, Viomycin formed higher number of H-bonds with SARS-CoV-2 Main Protease than its co-crystallised inhibitor compound N3. Molecular dynamics simulation further showed that Viomycin embedded deeply inside the binding pocket and formed robust binding with SARS-CoV-2 Main Protease. Therefore, we propose that Viomycin may act as a potential inhibitor of the Main Protease of SARS-CoV-2. Further optimisations with the drug may support the much-needed rapid response to mitigate the pandemic.Communicated by Ramaswamy H. Sarma.
Topics: Antiviral Agents; Coronavirus 3C Proteases; Drug Repositioning; Molecular Docking Simulation; Protease Inhibitors; SARS-CoV-2; Viomycin
PubMed: 32406317
DOI: 10.1080/07391102.2020.1768902 -
Proceedings of the National Academy of... May 2020Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal...
Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal subunit dissociation and trapping the ribosome in an intermediate state of intersubunit rotation. The mechanism by which viomycin stabilizes this state remains unexplained. To address this, we have determined cryo-EM and X-ray crystal structures of 70S ribosome complexes trapped in a rotated state by viomycin. The 3.8-Å resolution cryo-EM structure reveals a ribosome trapped in the hybrid state with 8.6° intersubunit rotation and 5.3° rotation of the 30S subunit head domain, bearing a single P/E state transfer RNA (tRNA). We identify five different binding sites for viomycin, four of which have not been previously described. To resolve the details of their binding interactions, we solved the 3.1-Å crystal structure of a viomycin-bound ribosome complex, revealing that all five viomycins bind to ribosomal RNA. One of these (Vio1) corresponds to the single viomycin that was previously identified in a complex with a nonrotated classical-state ribosome. Three of the newly observed binding sites (Vio3, Vio4, and Vio5) are clustered at intersubunit bridges, consistent with the ability of viomycin to inhibit subunit dissociation. We propose that one or more of these same three viomycins induce intersubunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23S rRNA, thus, blocking conformational changes associated with molecular movements that are required for translocation.
Topics: Anti-Bacterial Agents; Crystallography, X-Ray; Escherichia coli; Models, Molecular; Molecular Conformation; Protein Binding; Protein Biosynthesis; RNA, Messenger; RNA, Ribosomal; RNA, Transfer; Ribosomal Proteins; Ribosomes; Viomycin
PubMed: 32341159
DOI: 10.1073/pnas.2002888117