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Tuberactinomycin antibiotics: Biosynthesis, anti-mycobacterial action, and mechanisms of resistance.Frontiers in Microbiology 2022The tuberactinomycins are a family of cyclic peptide ribosome-targeting antibiotics with a long history of use as essential second-line treatments for drug-resistant... (Review)
Review
The tuberactinomycins are a family of cyclic peptide ribosome-targeting antibiotics with a long history of use as essential second-line treatments for drug-resistant tuberculosis. Beginning with the identification of viomycin in the early 1950s, this mini-review briefly describes tuberactinomycin structures and biosynthesis, as well as their past and present application in the treatment of tuberculosis caused by infection with . More recent studies are also discussed that have revealed details of tuberactinomycin action on the ribosome as well as resistance mechanisms that have emerged since their introduction into the clinic. Finally, future applications of these drugs are considered in the context of their recent removal from the World Health Organization's List of Essential Medicines.
PubMed: 36033858
DOI: 10.3389/fmicb.2022.961921 -
ACS Chemical Biology Jan 2022Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from subspecies . The gene cluster for CMN biosynthesis has been identified and...
Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from subspecies . The gene cluster for CMN biosynthesis has been identified and sequenced, wherein the gene was annotated as a phosphotransferase likely engaging in self-resistance. Previous studies reported that Cph inactivates two CMNs, CMN IA and IIA, by phosphorylation. We, herein, report that (1) harboring the gene becomes resistant to both CMN IIA and IIB, (2) phylogenetic analysis regroups Cph to a new clade in the phosphotransferase protein family, (3) Cph shares a three-dimensional structure akin to the aminoglycoside phosphotransferases with a high binding affinity () to both CMN IIA and IIB at micromolar levels, and (4) Cph utilizes either ATP or GTP as a phosphate group donor transferring its γ-phosphate to the hydroxyl group of CMN IIA. Until now, Cph and Vph (viomycin phosphotransferase) are the only two known enzymes inactivating peptide-based antibiotics through phosphorylation. Our biochemical characterization and structural determination conclude that Cph confers the gene-carrying species resistance to CMN by means of either chemical modification or physical sequestration, a naturally manifested belt and braces strategy. These findings add a new chapter into the self-resistance of bioactive natural products, which is often overlooked while designing new bioactive molecules.
Topics: Actinobacteria; Antibiotics, Antitubercular; Bacterial Proteins; Capreomycin; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Models, Molecular; Molecular Structure; Phosphotransferases (Alcohol Group Acceptor); Phylogeny; Protein Conformation
PubMed: 34994196
DOI: 10.1021/acschembio.1c00799 -
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 -
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 -
Frontiers in Chemistry 2024Many enzymes in nature utilize a free arginine (L-Arg) amino acid to initiate the biosynthesis of natural products. Examples include nitric oxide synthases, which... (Review)
Review
Many enzymes in nature utilize a free arginine (L-Arg) amino acid to initiate the biosynthesis of natural products. Examples include nitric oxide synthases, which generate NO from L-Arg for blood pressure control, and various arginine hydroxylases involved in antibiotic biosynthesis. Among the groups of arginine hydroxylases, several enzymes utilize a nonheme iron(II) active site and let L-Arg react with dioxygen and -ketoglutarate to perform either C-hydroxylation, C-hydroxylation, C-hydroxylation, or C-C-desaturation. How these seemingly similar enzymes can react with high specificity and selectivity to form different products remains unknown. Over the past few years, our groups have investigated the mechanisms of L-Arg-activating nonheme iron dioxygenases, including the viomycin biosynthesis enzyme VioC, the naphthyridinomycin biosynthesis enzyme NapI, and the streptothricin biosynthesis enzyme OrfP, using computational approaches and applied molecular dynamics, quantum mechanics on cluster models, and quantum mechanics/molecular mechanics (QM/MM) approaches. These studies not only highlight the differences in substrate and oxidant binding and positioning but also emphasize on electronic and electrostatic differences in the substrate-binding pockets of the enzymes. In particular, due to charge differences in the active site structures, there are changes in the local electric field and electric dipole moment orientations that either strengthen or weaken specific substrate C-H bonds. The local field effects, therefore, influence and guide reaction selectivity and specificity and give the enzymes their unique reactivity patterns. Computational work using either QM/MM or density functional theory (DFT) on cluster models can provide valuable insights into catalytic reaction mechanisms and produce accurate and reliable data that can be used to engineer proteins and synthetic catalysts to perform novel reaction pathways.
PubMed: 38406558
DOI: 10.3389/fchem.2024.1365494 -
Frontiers in Microbiology 2022Resistance to tuberculosis (TB) drugs has become a major threat to global control efforts. Early case detection and drug susceptibility profiling of the infecting...
BACKGROUND
Resistance to tuberculosis (TB) drugs has become a major threat to global control efforts. Early case detection and drug susceptibility profiling of the infecting bacteria are essential for appropriate case management. The objective of this study was to determine the drug susceptibility profiles of difficult-to-treat (DTT) TB patients in Ghana.
METHODS
Sputum samples obtained from DTT-TB cases from health facilities across Ghana were processed for rapid diagnosis and detection of drug resistance using the Genotype MTBDR and Genotype MTBDR. from Hain Life science.
RESULTS
A total of 298 (90%) out of 331 sputum samples processed gave interpretable bands out of which 175 (58.7%) were resistant to at least one drug (ANY); 16.8% (50/298) were isoniazid-mono-resistant (INH), 16.8% (50/298) were rifampicin-mono-resistant (RIF), and 25.2% (75/298) were MDR. 24 (13.7%) of the ANY were additionally resistant to at least one second line drug: 7.4% (2 RIF, 1 INH, and 10 MDR samples) resistant to only FQs and 2.3% (2 RIF, 1 INH, and 1 MDR samples) resistant to AMG drugs kanamycin (KAN), amikacin (AMK), capreomycin (CAP), and viomycin (VIO). Additionally, there were 4.0% (5 RIF and 2 MDR samples) resistant to both FQs and AMGs. 81 (65.6%) out of 125 INH-resistant samples including INH and MDR had -mutations (MT) whereas 15 (12%) had -MT. The remaining 28 (22.4%) had both and MT. All the 19 FQ-resistant samples were mutants whereas the 10 AMGs were (3), (3) as well as , and co-mutants (4). Except for the seven pre-XDR samples, no sample had MT.
CONCLUSION
The detection of several pre-XDR TB cases in Ghana calls for intensified drug resistance surveillance and monitoring of TB patients to, respectively, ensure early diagnosis and treatment compliance.
PubMed: 36713197
DOI: 10.3389/fmicb.2022.1069292 -
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 -
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 -
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 -
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