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1.
mBio ; 12(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33402533

RESUMO

Despite dogma suggesting that lipopolysaccharide/lipooligosaccharide (LOS) was essential for viability of Gram-negative bacteria, several Acinetobacter baumannii clinical isolates produced LOS- colonies after colistin selection. Inactivation of the conserved class A penicillin-binding protein, PBP1A, was a compensatory mutation that supported isolation of LOS- A. baumannii, but the impact of PBP1A mutation was not characterized. Here, we show that the absence of PBP1A causes septation defects and that these, together with ld-transpeptidase activity, support isolation of LOS- A. baumannii PBP1A contributes to proper cell division in A. baumannii, and its absence induced cell chaining. Only isolates producing three or more septa supported selection of colistin-resistant LOS- A. baumannii PBP1A was enriched at the midcell, where the divisome complex facilitates daughter cell formation, and its localization was dependent on glycosyltransferase activity. Transposon mutagenesis showed that genes encoding two putative ld-transpeptidases (LdtJ and LdtK) became essential in the PBP1A mutant. Both LdtJ and LdtK were required for selection of LOS- A. baumannii, but each had distinct enzymatic activities in the cell. Together, these findings demonstrate that defects in PBP1A glycosyltransferase activity and ld-transpeptidase activity remodel the cell envelope to support selection of colistin-resistant LOS- A. baumannii IMPORTANCE The increasing prevalence of antibiotic treatment failure associated with Gram-negative bacterial infections highlights an urgent need to develop new alternative therapeutic strategies. The last-line antimicrobial colistin (polymyxin E) targets the ubiquitous outer membrane lipopolysaccharide (LPS)/LOS membrane anchor, lipid A, which is essential for viability of most diderms. However, several LOS- Acinetobacter baumannii clinical isolates were recovered after colistin selection, suggesting a conserved resistance mechanism. Here, we characterized a role for penicillin-binding protein 1A in A. baumannii septation and intrinsic ß-lactam susceptibility. We also showed that defects in PBP1A glycosyltransferase activity and ld-transpeptidase activity support isolation of colistin-resistant LOS- A. baumannii.


Assuntos
Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Lipopolissacarídeos/deficiência , Proteínas de Ligação às Penicilinas/metabolismo , Peptidil Transferases/metabolismo , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Lipídeo A/metabolismo , Lipopolissacarídeos/genética , Testes de Sensibilidade Microbiana , Peptidoglicano Glicosiltransferase
2.
Nucleic Acids Res ; 49(1): 206-220, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330942

RESUMO

Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5' region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.


Assuntos
Chaperonas Moleculares/fisiologia , Complexos Multiproteicos/fisiologia , Elongação Traducional da Cadeia Peptídica/fisiologia , Dobramento de Proteína , Proteostase/fisiologia , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Alelos , Mutação com Perda de Função , Chaperonas Moleculares/genética , Mutação de Sentido Incorreto , Peptidil Transferases/fisiologia , Mutação Puntual , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/fisiologia , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
3.
Chemistry ; 27(10): 3542-3551, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33336443

RESUMO

The carbapenem class of ß-lactams has been optimized against Gram-negative bacteria producing extended-spectrum ß-lactamases by introducing substituents at position C2. Carbapenems are currently investigated for the treatment of tuberculosis as these drugs are potent covalent inhibitors of l,d-transpeptidases involved in mycobacterial cell wall assembly. The optimization of carbapenems for inactivation of these unusual targets is sought herein by exploiting the nucleophilicity of the C8 hydroxyl group to introduce chemical diversity. As ß-lactams are structure analogs of peptidoglycan precursors, the substituents were chosen to increase similarity between the drug and the substrate. Fourteen peptido-carbapenems were efficiently synthesized. They were more effective than the reference drug, meropenem, owing to the positive impact of a phenethylthio substituent introduced at position C2 but the peptidomimetics added at position C8 did not further improve the activity. Thus, position C8 can be modified to modulate the pharmacokinetic properties of highly efficient carbapenems.


Assuntos
Carbapenêmicos/química , Antibacterianos/farmacologia , Parede Celular , Meropeném , Peptidoglicano , Peptidil Transferases
4.
Nat Commun ; 11(1): 2900, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518240

RESUMO

5S rRNA is an indispensable component of cytoplasmic ribosomes in all species. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether 5S rRNA autonomy is critical for ribosome function and cell survival. By linking circularly permutated 5S rRNA with 23S rRNA we generated a bacterial strain devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is dispensable for cell growth under standard conditions and is unlikely to have essential functions outside the ribosome. The fully assembled ribosomes carrying 23S-5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits unable to form stable 70S ribosomes. Cryo-EM analysis revealed a malformed peptidyl transferase center in the misassembled 50S subunits. Our results argue that the autonomy of 5S rRNA is preserved due to its role in ribosome biogenesis.


Assuntos
RNA Ribossômico 5S/metabolismo , Ribossomos/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação da Expressão Gênica , Engenharia Genética , Mutação , Conformação de Ácido Nucleico , Peptidil Transferases/metabolismo , RNA Bacteriano , RNA Ribossômico 23S/metabolismo , Recombinases Rec A/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo
5.
Nat Commun ; 11(1): 1858, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313034

RESUMO

Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.


Assuntos
Bactérias/metabolismo , Engenharia de Proteínas/métodos , Ribossomos/química , Biologia Sintética/métodos , Alelos , Sistema Livre de Células , Cristalografia por Raios X , Modelos Moleculares , Modelos Teóricos , Conformação Molecular , Mutação , Peptídeos/química , Peptidil Transferases/química , Plasmídeos/genética , Biossíntese de Proteínas , Proteoma , RNA Mensageiro/genética , RNA Ribossômico/genética , RNA Ribossômico 23S/genética , Thermus thermophilus/química
6.
Mol Cell ; 78(4): 700-713.e7, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32289254

RESUMO

Impairment of ribosome function activates the MAPKKK ZAK, leading to activation of mitogen-activated protein (MAP) kinases p38 and JNK and inflammatory signaling. The mechanistic basis for activation of this ribotoxic stress response (RSR) remains completely obscure. We show that the long isoform of ZAK (ZAKα) directly associates with ribosomes by inserting its flexible C terminus into the ribosomal intersubunit space. Here, ZAKα binds helix 14 of 18S ribosomal RNA (rRNA). An adjacent domain in ZAKα also probes the ribosome, and together, these sensor domains are critically required for RSR activation after inhibition of both the E-site, the peptidyl transferase center (PTC), and ribotoxin action. Finally, we show that ablation of the RSR response leads to organismal phenotypes and decreased lifespan in the nematode Caenorhabditis elegans (C. elegans). Our findings yield mechanistic insight into how cells detect ribotoxic stress and provide experimental in vivo evidence for its physiological importance.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , MAP Quinase Quinase Quinases/metabolismo , Peptidil Transferases/metabolismo , RNA Ribossômico 18S/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Ativação Enzimática , Células HeLa , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/genética , Conformação Proteica , Domínios Proteicos , RNA Ribossômico 18S/genética , Homologia de Sequência , Transdução de Sinais
7.
Eur J Med Chem ; 194: 112262, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32248005

RESUMO

The enzymes involved in bacterial cell wall synthesis are established antibiotic targets, and continue to be a central focus for antibiotic development. Bacterial penicillin-binding proteins (and, in some bacteria, l,d-transpeptidases) form essential peptide cross-links in the cell wall. Although the ß-lactam class of antibiotics target these enzymes, bacterial resistance threatens their clinical use, and there is an urgent unmet need for new antibiotics. However, the search for new antibiotics targeting the bacterial cell wall is hindered by a number of obstacles associated with screening the enzymes involved in peptidoglycan synthesis. This review describes recent approaches for measuring the activity and inhibition of penicillin-binding proteins and l,d-transpeptidases, highlighting strategies that are poised to serve as valuable tools for high-throughput screening of transpeptidase inhibitors, supporting the development of new antibiotics.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Descoberta de Drogas , Peptidil Transferases/antagonistas & inibidores , Antibacterianos/química , Bactérias/metabolismo , Parede Celular/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Proteínas de Ligação às Penicilinas/metabolismo , Peptidil Transferases/metabolismo
8.
Nat Commun ; 11(1): 1108, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111839

RESUMO

Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7 × 107 member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4 × 103 rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.


Assuntos
Ribossomos/genética , Ribossomos/metabolismo , Antibacterianos/farmacologia , Clindamicina/farmacologia , Evolução Molecular Direcionada , Farmacorresistência Bacteriana/genética , Epistasia Genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca Gênica , Genótipo , Mutação , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Ribossômico/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/efeitos dos fármacos , Biologia Sintética
9.
Nucleic Acids Res ; 48(5): 2777-2789, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32009164

RESUMO

The synthetic capability of the Escherichia coli ribosome has attracted efforts to repurpose it for novel functions, such as the synthesis of polymers containing non-natural building blocks. However, efforts to repurpose ribosomes are limited by the lack of complete peptidyl transferase center (PTC) active site mutational analyses to inform design. To address this limitation, we leverage an in vitro ribosome synthesis platform to build and test every possible single nucleotide mutation within the PTC-ring, A-loop and P-loop, 180 total point mutations. These mutant ribosomes were characterized by assessing bulk protein synthesis kinetics, readthrough, assembly, and structure mapping. Despite the highly-conserved nature of the PTC, we found that >85% of the PTC nucleotides possess mutational flexibility. Our work represents a comprehensive single-point mutant characterization and mapping of the 70S ribosome's active site. We anticipate that it will facilitate structure-function relationships within the ribosome and make possible new synthetic biology applications.


Assuntos
Domínio Catalítico , Escherichia coli/metabolismo , Mutação/genética , Ribossomos/química , Ribossomos/genética , Códon/genética , Modelos Moleculares , Peptidil Transferases/metabolismo , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Ribossômico/metabolismo
10.
Nucleic Acids Res ; 48(5): 2723-2732, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31989172

RESUMO

Post-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides' positions, a task that has previously been accomplished by X-ray crystallography. Here, we present a cryo-electron microscopy (cryo-EM) structure of the Escherichia coli 50S subunit at an average resolution of 2.2 Å as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for assessment of cryo-EM map quality. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible.


Assuntos
Microscopia Crioeletrônica , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Nucleotídeos/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Modelos Moleculares , Peptídeos/metabolismo , Peptidil Transferases/metabolismo , Reprodutibilidade dos Testes , Solventes , Eletricidade Estática
11.
J Mol Biol ; 432(4): 978-990, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31877323

RESUMO

RNA helicases play various roles in ribosome biogenesis depending on the ribosome assembly pathway and stress state of the cell. However, it is unclear how most RNA helicases interact with ribosome assembly intermediates or participate in other cell processes to regulate ribosome assembly. SrmB is a DEAD-box helicase that acts early in the ribosome assembly process, although very little is known about its mechanism of action. Here, we use a combined quantitative mass spectrometry/cryo-electron microscopy approach to detail the protein inventory, rRNA modification state, and structures of 40S ribosomal intermediates that form upon SrmB deletion. We show that the binding site of SrmB is unperturbed by SrmB deletion, but the peptidyl transferase center, the uL7/12 stalk, and 30S contact sites all show severe assembly defects. Taking into account existing data on SrmB function and the experiments presented here, we propose several mechanisms by which SrmB could guide assembling particles from kinetic traps to competent subunits during the 50S ribosome assembly process.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Escherichia coli/metabolismo , Sítios de Ligação/genética , Microscopia Crioeletrônica , RNA Helicases DEAD-box/genética , Proteínas de Escherichia coli/genética , Espectrometria de Massas , Mutação/genética , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Subunidades Ribossômicas Maiores de Arqueas/genética , Subunidades Ribossômicas Maiores de Arqueas/metabolismo , Subunidades Ribossômicas Maiores de Arqueas/ultraestrutura , Ribossomos/metabolismo , Ribossomos/ultraestrutura
12.
Biochem Biophys Res Commun ; 523(1): 6-9, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-31822344

RESUMO

Tuberculosis has attracted increased attention worldwide due to its high morality and its resistance to treatment with traditional antibacterial drugs. The l,d-transpeptidase LdtMt2 confers resistance to traditional ß-lactams and is considered a target for anti-Tuberculosis treatment. Carbapenems are proposed to inhibit Mycobacterium tuberculosis by repressing the activity of LdtMt2. The interaction mechanisms between LdtMt2 and carbapenems have been revealed by LdtMt2-carbapenem adduct structures along with various biochemical assays. Interestingly, the lack of the 1-ß-methyl group in imipenem may be related to its high binding ability to LdtMt2. However, there is limited evidence on the interaction mode of LdtMt2 and panipenem, another carbapenem lacking the 1-ß-methyl group. Herein, we identified the biochemical features of panipenem binding to LdtMt2. We further suggest that the presence of the 1-ß-methyl group in carbapenems is indeed related to the ligand affinity of LdtMt2 and that the presence of the Y308 and Y318 residues in LdtMt2 stabilized the conformation of the LdtMt2-carbepenem adduct. Our research provides a structural basis for the development of novel carbapenems against L,D-transpeptidases.


Assuntos
Inibidores Enzimáticos/farmacologia , Peptidil Transferases/antagonistas & inibidores , Tienamicinas/farmacologia , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Peptidil Transferases/química , Peptidil Transferases/metabolismo , Tienamicinas/química
13.
Curr Med Chem ; 27(19): 3250-3267, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30501595

RESUMO

Peptidoglycan, the exoskeleton of bacterial cell and an essential barrier that protects the cell, is synthesized by a pathway where the final steps are catalysed by transpeptidases. Knowledge of the structure and function of these vital enzymes that generate this macromolecule in M. tuberculosis could facilitate the development of potent lead compounds against tuberculosis. This review summarizes the experimental and computational studies to date on these aspects of transpeptidases in M. tuberculosis that have been identified and validated. The reported structures of L,D- and D,D-transpeptidases, as well as their functionalities, are reviewed and the proposed enzymatic mechanisms for L,D-transpeptidases are summarized. In addition, we provide bioactivities of known tuberculosis drugs against these enzymes based on both experimental and computational approaches. Advancing knowledge about these prominent targets supports the development of new drugs with novel inhibition mechanisms overcoming the current need for new drugs against tuberculosis.


Assuntos
Mycobacterium tuberculosis , Proteínas de Bactérias , Parede Celular , Peptidoglicano , Peptidil Transferases
14.
Cell Chem Biol ; 26(12): 1639-1641, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31680066

RESUMO

The possible mechanism(s) by which ribosomes make peptide bonds during protein synthesis have been explored for decades. Yet, there is no agreement on how the catalytic site, the peptidyl transferase center (PTC), promotes this reaction. Here, we discuss the results of recent investigations of translation with D amino acids that provide fresh insights into that longstanding question.


Assuntos
Aminoácidos/metabolismo , Peptidil Transferases/metabolismo , Ribossomos/metabolismo , Aminoácidos/química , Biocatálise , Domínio Catalítico , Peptidil Transferases/química , Biossíntese de Proteínas , RNA de Transferência/química , RNA de Transferência/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo
15.
Int J Mol Sci ; 20(21)2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31671806

RESUMO

The Enzyme Classification (EC) number is a numerical classification scheme for enzymes, established using the chemical reactions they catalyze. This classification is based on the recommendation of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. Six enzyme classes were recognised in the first Enzyme Classification and Nomenclature List, reported by the International Union of Biochemistry in 1961. However, a new enzyme group was recently added as the six existing EC classes could not describe enzymes involved in the movement of ions or molecules across membranes. Such enzymes are now classified in the new EC class of translocases (EC 7). Several computational methods have been developed in order to predict the EC number. However, due to this new change, all such methods are now outdated and need updating. In this work, we developed a new multi-task quantitative structure-activity relationship (QSAR) method aimed at predicting all 7 EC classes and subclasses. In so doing, we developed an alignment-free model based on artificial neural networks that proved to be very successful.


Assuntos
Enzimas/química , Enzimas/classificação , Relação Quantitativa Estrutura-Atividade , Algoritmos , Biologia Computacional/métodos , Bases de Dados Factuais , Enzimas/metabolismo , Modelos Lineares , Aprendizado de Máquina , Dinâmica não Linear , Peptidil Transferases , Proteínas/química , Proteínas/genética , Sensibilidade e Especificidade
16.
ACS Chem Biol ; 14(12): 2745-2756, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31743648

RESUMO

Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.


Assuntos
Aminoácidos/metabolismo , Sondas Moleculares/metabolismo , Peptidoglicano/biossíntese , Bacillus subtilis/metabolismo , Parede Celular/metabolismo , Citoplasma/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Peptidil Transferases/metabolismo
17.
J Mol Model ; 25(11): 328, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31656981

RESUMO

Virtual screening is a useful in silico approach to identify potential leads against various targets. It is known that carbapenems (doripenem and faropenem) do not show any reasonable inhibitory activities against L,D-transpeptidase 5 (LdtMt5) and also an adduct of meropenem exhibited slow acylation. Since these drugs are active against L,D-transpeptidase 2 (LdtMt2), understanding the differences between these two enzymes is essential. In this study, a ligand-based virtual screening of 12,766 compounds followed by molecular dynamics (MD) simulations was applied to identify potential leads against LdtMt5. To further validate the obtained virtual screening ranking for LdtMt5, we screened the same libraries of compounds against LdtMt2 which had more experimetal and calculated binding energies reported. The observed consistency between the binding affinities of LdtMt2 validates the obtained virtual screening binding scores for LdtMt5. We subjected 37 compounds with docking scores ranging from - 7.2 to - 9.9 kcal mol-1 obtained from virtual screening for further MD analysis. A set of compounds (n = 12) from four antibiotic classes with ≤ - 30 kcal mol-1 molecular mechanics/generalized born surface area (MM-GBSA) binding free energies (ΔGbind) was characterized. A final set of that, all ß-lactams (n = 4), was considered. The outcome of this study provides insight into the design of potential novel leads for LdtMt5. Graphical abstract.


Assuntos
Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Antibacterianos/farmacologia , Ligantes , Meropeném/farmacologia , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Peptidil Transferases/antagonistas & inibidores , Ligação Proteica/efeitos dos fármacos
18.
Microb Genom ; 5(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31609685

RESUMO

Penicillin-non-susceptible Streptococcus pneumoniae (PNSP) were first detected in the 1960s, and are now common worldwide, predominantly through the international spread of a limited number of strains. Extant PNSP are characterized by mosaic pbp2x, pbp2b and pbp1a genes generated by interspecies recombinations, with the extent of these alterations determining the range and concentrations of ß-lactams to which the genotype is non-susceptible. The complexity of the genetics underlying these phenotypes has been the subject of both molecular microbiology and genome-wide association and epistasis analyses. Such studies can aid our understanding of PNSP evolution and help improve the already highly-performing bioinformatic methods capable of identifying PNSP from genomic surveillance data.


Assuntos
Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Resistência às Penicilinas/genética , Proteínas de Ligação às Penicilinas/genética , Peptidil Transferases/genética , Infecções Pneumocócicas , Streptococcus pneumoniae/genética , Genoma Bacteriano/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Penicilinas/metabolismo , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia
19.
ACS Infect Dis ; 5(12): 2047-2054, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31597040

RESUMO

The genomes of diverse mycobacterial species encode multiple proteins with the canonical l,d-transpeptidase (Ldt) sequence motif. The reason for this apparent redundancy is not well understood, but evidence suggests paralogous Ldts may serve niche roles in maintaining and/or remodeling mycobacterial peptidoglycan. We examined 323 mycobacterial Ldts and determined these enzymes cluster into six clades. We identified a variably represented yet distinct Ldt class (class 6) containing Mycobacterium smegmatis (Msm) LdtF and built a homology model of Msm LdtF toward elucidating class 6 structural and functional differences. We report class 6 Ldts have structurally divergent catalytic domains containing a 10-residue insertion near the active site and additionally determined that meropenem preferentially acylates LdtF. Our data demonstrate an evolutionary basis for mycobacterial Ldt multiplicity that lends support to the idea that paralogous Ldts serve nonredundant roles in vivo and suggests class 6 Ldts can be selectively targeted by specific carbapenem antibiotics.


Assuntos
Antibacterianos/farmacologia , Meropeném/farmacologia , Mycobacterium/enzimologia , Peptidil Transferases/química , Peptidil Transferases/classificação , Acilação , Motivos de Aminoácidos , Domínio Catalítico , Evolução Molecular , Modelos Moleculares , Família Multigênica , Mycobacterium/efeitos dos fármacos , Mycobacterium/genética , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Filogenia , Homologia de Sequência de Aminoácidos
20.
Chem Commun (Camb) ; 55(69): 10214-10217, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31380528

RESUMO

The l,d-transpeptidases (Ldts) are promising antibiotic targets for treating tuberculosis. We report screening of cysteine-reactive inhibitors against LdtMt2 from Mycobacterium tuberculosis. Structural studies on LdtMt2 with potent inhibitor ebselen reveal opening of the benzisoselenazolone ring by a nucleophilic cysteine, forming a complex involving extensive hydrophobic interactions with a substrate-binding loop.


Assuntos
Azóis/química , Azóis/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Compostos Organosselênicos/química , Compostos Organosselênicos/farmacologia , Peptidil Transferases/antagonistas & inibidores , Antituberculosos/química , Antituberculosos/farmacologia , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Cisteína/metabolismo , Humanos , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Peptidil Transferases/metabolismo , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
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