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1.
Nucleic Acids Res ; 49(15): 8777-8784, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365509

RESUMO

Transcribing RNA polymerase (RNAP) can fall into backtracking, phenomenon when the 3' end of the transcript disengages from the template DNA. Backtracking is caused by sequences of the nucleic acids or by misincorporation of erroneous nucleotides. To resume productive elongation backtracked complexes have to be resolved through hydrolysis of RNA. There is currently no consensus on the mechanism of catalysis of this reaction by Escherichia coli RNAP. Here we used Salinamide A, that we found inhibits RNAP catalytic domain Trigger Loop (TL), to show that the TL is required for RNA cleavage during proofreading of misincorporation events but plays little role during cleavage in sequence-dependent backtracked complexes. Results reveal that backtracking caused by misincorporation is distinct from sequence-dependent backtracking, resulting in different conformations of the 3' end of RNA within the active center. We show that the TL is required to transfer the 3' end of misincorporated transcript from cleavage-inefficient 'misincorporation site' into the cleavage-efficient 'backtracked site', where hydrolysis takes place via transcript-assisted catalysis and is largely independent of the TL. These findings resolve the controversy surrounding mechanism of RNA hydrolysis by E. coli RNA polymerase and indicate that the TL role in RNA cleavage has diverged among bacteria.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , RNA Mensageiro/metabolismo , Elongação da Transcrição Genética , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrólise , Clivagem do RNA
2.
J Chem Phys ; 154(19): 195103, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34240890

RESUMO

Interactions among ions and their specific interactions with macromolecular solutes are known to play a central role in biomolecular stability. However, similar effects in the conformational stability of protein loops that play functional roles, such as binding ligands, proteins, and DNA/RNA molecules, remain relatively unexplored. A well-characterized enzyme that has such a functional loop is Escherichia coli dihydrofolate reductase (ecDHFR), whose so-called M20 loop has been observed in three ordered conformations in crystal structures. To explore how solution ionic strengths may affect the M20 loop conformation, we proposed a reaction coordinate that could quantitatively describe the loop conformation and used it to classify the loop conformations in representative ecDHFR x-ray structures crystallized in varying ionic strengths. The Protein Data Bank survey indicates that at ionic strengths (I) below the intracellular ion concentration-derived ionic strength in E. coli (I ≤ 0.237M), the ecDHFR M20 loop tends to adopt open/closed conformations, and rarely an occluded loop state, but when I is >0.237M, the loop tends to adopt closed/occluded conformations. Distance-dependent electrostatic potentials around the most mobile M20 loop region from molecular dynamics simulations of ecDHFR in equilibrated CaCl2 solutions of varying ionic strengths show that high ionic strengths (I = 0.75/1.5M) can preferentially stabilize the loop in closed/occluded conformations. These results nicely correlate with conformations derived from ecDHFR structures crystallized in varying ionic strengths. Altogether, our results suggest caution in linking M20 loop conformations derived from crystal structures solved at ionic strengths beyond that tolerated by E. coli to the ecDHFR function.


Assuntos
Cloreto de Cálcio/química , Escherichia coli/enzimologia , Tetra-Hidrofolato Desidrogenase/química , Simulação de Dinâmica Molecular , Concentração Osmolar , Conformação Proteica , Soluções , Tetra-Hidrofolato Desidrogenase/metabolismo
3.
Methods Mol Biol ; 2341: 37-44, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264459

RESUMO

The lacZ gene and corresponding ß-galactosidase enzyme has been a mainstay for bacterial reporter systems for decades. We have used this versatile reporter to analyze expression profiles from strains grown both on solid media and from broth culture. The standard broth protocol can also be adapted for a 96-well plate to allow high-throughput screening of promoter reporter constructs under a variety of conditions. Furthermore, codon-optimization of the E. coli lacZ gene has greatly improved activity levels of ß-galactosidase in S. aureus, facilitating improved sensitivity for screening assays, detection of low-activity promoters, and use of small sample volumes. In this chapter, details are provided for both standard and high-throughput quantitative assays that we have routinely used for S. aureus transcriptional profiling.


Assuntos
Toxinas Bacterianas/genética , Escherichia coli/enzimologia , Proteínas Hemolisinas/genética , Ensaios de Triagem em Larga Escala/métodos , Staphylococcus aureus/genética , beta-Galactosidase/genética , Proteínas de Bactérias/genética , Composição de Bases , Códon , Escherichia coli/genética , Expressão Gênica , Genes Reporter , Óperon Lac , Regiões Promotoras Genéticas
4.
J Chem Phys ; 155(3): 035101, 2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34293874

RESUMO

Escherichia coli adenylate kinase (AK) is composed of CORE domain and two branch domains: LID and AMP-binding domain (AMPbd). AK exhibits considerable allostery in a reversible phosphoryl transfer reaction, which is largely attributed to the relative motion of LID and AMPbd with respect to CORE. Such an allosteric conformational change is also evident in the absence of ligands. Recent studies showed that the mutations in branch domains can adjust dynamic allostery and alter the substrate affinity and enzyme activity. In this work, we use all-atom molecular dynamics simulation to study the impacts of mutations in branch domains on AK's dynamic allostery by comparing two double mutants, i.e., LID mutant (Val135Gly, Val142Gly) and AMPbd mutant (Ala37Gly, Ala55Gly), with wild-type. Two mutants undergo considerable conformational fluctuation and exhibit deviation from the initially extended apo state to more compact structures. The LID domain in the LID mutant adjusts its relative position and firmly adheres to CORE. More strikingly, AMPbd mutations affect the relative positions of both the AMPbd domain and remote LID domain. Both domains undergo considerable movement, especially the inherent hinge swing motion of the flexible LID domain. In both mutants, the mutations can enhance the inter-domain interaction. The results about the conformation change of AK in both mutants are in line with the experiment of AK's affinity and activity. As revealed by our findings, the flexibility of branch domains and their inherent motions, especially LID domain, is highly relevant to dynamic allostery in the AK system.


Assuntos
Adenilato Quinase/metabolismo , Mutação , Adenilato Quinase/química , Adenilato Quinase/genética , Regulação Alostérica , Escherichia coli/enzimologia , Conformação Proteica , Domínios Proteicos
5.
Molecules ; 26(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34200016

RESUMO

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.


Assuntos
Antibacterianos/farmacologia , Endorribonucleases/genética , Escherichia coli/enzimologia , Oligonucleotídeos/farmacologia , Sistema Livre de Células , Endorribonucleases/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Oligonucleotídeos/síntese química , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , RNA Mensageiro/antagonistas & inibidores
6.
Nucleic Acids Res ; 49(11): 6389-6398, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34086932

RESUMO

Biogenesis of ribosomal subunits involves enzymatic modifications of rRNA that fine-tune functionally important regions. The universally conserved prokaryotic dimethyltransferase KsgA sequentially modifies two universally conserved adenosine residues in helix 45 of the small ribosomal subunit rRNA, which is in proximity of the decoding site. Here we present the cryo-EM structure of Escherichia coli KsgA bound to an E. coli 30S at a resolution of 3.1 Å. The high-resolution structure reveals how KsgA recognizes immature rRNA and binds helix 45 in a conformation where one of the substrate nucleotides is flipped-out into the active site. We suggest that successive processing of two adjacent nucleotides involves base-flipping of the rRNA, which allows modification of the second substrate nucleotide without dissociation of the enzyme. Since KsgA is homologous to the essential eukaryotic methyltransferase Dim1 involved in 40S maturation, these results have also implications for understanding eukaryotic ribosome maturation.


Assuntos
Adenosina/metabolismo , Escherichia coli/enzimologia , Metiltransferases/química , Adenosina/química , Microscopia Crioeletrônica , Metiltransferases/metabolismo , Modelos Moleculares , Conformação Proteica , Subunidades Ribossômicas Menores de Bactérias/química , Especificidade por Substrato
7.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34074047

RESUMO

In this review, we chart the major milestones in the research progress on the DyP-type peroxidase family over the past decade. Though mainly distributed among bacteria and fungi, this family actually exhibits more widespread diversity. Advanced tertiary structural analyses have revealed common and different features among members of this family. Notably, the catalytic cycle for the peroxidase activity of DyP-type peroxidases appears to be different from that of other ubiquitous heme peroxidases. DyP-type peroxidases have also been reported to possess activities in addition to peroxidase function, including hydrolase or oxidase activity. They also show various cellular distributions, functioning not only inside cells but also outside of cells. Some are also cargo proteins of encapsulin. Unique, noteworthy functions include a key role in life-cycle switching in Streptomyces and the operation of an iron transport system in Staphylococcus aureus, Bacillus subtilis and Escherichia coli. We also present several probable physiological roles of DyP-type peroxidases that reflect the widespread distribution and function of these enzymes. Lignin degradation is the most common function attributed to DyP-type peroxidases, but their activity is not high compared with that of standard lignin-degrading enzymes. From an environmental standpoint, degradation of natural antifungal anthraquinone compounds is a specific focus of DyP-type peroxidase research. Considered in its totality, the DyP-type peroxidase family offers a rich source of diverse and attractive materials for research scientists.


Assuntos
Antraquinonas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Lignina/metabolismo , Peroxidases/química , Peroxidases/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Catálise , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Fungos/crescimento & desenvolvimento , Ferro/metabolismo , Oxirredução , Filogenia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismo , Streptomyces/enzimologia , Streptomyces/metabolismo
8.
Int J Biol Macromol ; 182: 2130-2143, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34087308

RESUMO

For centuries, dietary ginger has been known for its antioxidant, anticancer, and antibacterial properties. In the current study, we examined the link between antibacterial properties of 7 dietary ginger phenolics (DGPs)-gingerenone A, 6-gingerol, 8-gingerol, 10-gingerol, paradol, 6-shogaol, and zingerone-and inhibition of bacterial ATP synthase. DGPs caused complete (100%) inhibition of wild-type Escherichia coli membrane-bound F1Fo ATP synthase, but partial and variable (0%-87%) inhibition of phytochemical binding site mutant enzymes αR283D, αE284R, ßV265Q, and γT273A. The mutant enzyme ATPase activity was 16-fold to 100-fold lower than that of the wild-type enzyme. The growth of wild-type, null, and mutant strains in the presence of the 7 DGPs were abrogated to variable degrees on limiting glucose and succinate media. DGPs-caused variable inhibitory profiles of wild-type and mutant ATP synthase confirm that residues of α-, ß-, and γ-subunits are involved in the formation of phytochemical binding site. The variable degree of growth in the presence of DGPs also indicates the possibility of molecular targets other than ATP synthase. Our results establish that antibacterial properties of DGPs can be linked to the binding and inhibition of bacterial ATP synthase. Therefore, bacterial ATP synthase is a valuable molecular target for DGPs.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Dieta , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Gengibre/química , Fenóis/farmacologia , Adenosina Trifosfatases/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Glucose/metabolismo , Mutação/genética , Fenóis/química , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Ácido Succínico/metabolismo
9.
J Biol Chem ; 297(1): 100812, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34023383

RESUMO

In vitro studies of transcription frequently require the preparation of defined elongation complexes. Defined transcription elongation complexes (TECs) are typically prepared by constructing an artificial transcription bubble from synthetic oligonucleotides and RNA polymerase. This approach is optimal for diverse applications but is sensitive to nucleic acid length and sequence and is not compatible with systems where promoter-directed initiation or extensive transcription elongation is crucial. To complement scaffold-directed approaches for TEC assembly, I have developed a method for preparing promoter-initiated Escherichia coli TECs using a purification strategy called selective photoelution. This approach combines TEC-dependent sequestration of a biotin-triethylene glycol transcription stall site with photoreversible DNA immobilization to enrich TECs from an in vitro transcription reaction. I show that selective photoelution can be used to purify TECs that contain a 273-bp DNA template and 194-nt structured RNA. Selective photoelution is a straightforward and robust procedure that, in the systems assessed here, generates precisely positioned TECs with >95% purity and >30% yield. TECs prepared by selective photoelution can contain complex nucleic acid sequences and will therefore likely be useful for investigating RNA structure and function in the context of RNA polymerases.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Luz , Fenômenos Magnéticos , Microesferas , Elongação da Transcrição Genética , Pareamento de Bases , Biotina/química , Regiões Promotoras Genéticas , RNA/química , Estreptavidina/química
10.
Euro Surveill ; 26(21)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34047273

RESUMO

The hospital water environment, including the wastewater drainage system, is increasingly reported as a potential reservoir for carbapenemase-producing Enterobacterales (CPE). We investigated a persistent outbreak of OXA-48 CPE (primarily Citrobacter freundii) in a haematological ward of a French teaching hospital by epidemiological, microbiological and environmental methods. Between January 2016 and June 2019, we detected 37 new OXA-48 CPE-colonised and/or ­infected patients in the haematological ward. In October 2017, a unit dedicated to CPE-colonised and/or ­infected patients was created. Eleven additional sporadic acquisitions were identified after this date without any obvious epidemiological link between patients, except in one case. Environmental investigations of the haematological ward (June-August 2018) identified seven of 74 toilets and one of 39 drains positive for OXA-48 CPE (seven C. freundii, one Enterobacter sakazakii, one Escherichia coli). Whole genome comparisons identified a clonal dissemination of OXA-48-producing C. freundii from the hospital environment to patients. In addition to strict routine infection control measures, an intensive cleaning programme was performed (descaling and bleaching) and all toilet bowls and tanks were changed. These additional measures helped to contain the outbreak. This study highlights that toilets can be a possible source of transmission of OXA-48 CPE.


Assuntos
Infecção Hospitalar/microbiologia , Surtos de Doenças , Infecções por Enterobacteriaceae/microbiologia , Toaletes , Proteínas de Bactérias , Citrobacter freundii/enzimologia , Cronobacter sakazakii/enzimologia , Reservatórios de Doenças/microbiologia , Escherichia coli/enzimologia , França/epidemiologia , Hospitais , Humanos , Controle de Infecções , Microbiologia da Água , beta-Lactamases/genética
11.
Arch Biochem Biophys ; 707: 108899, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-33991499

RESUMO

ATP synthase is essential in aerobic energy metabolism, and the rotary catalytic mechanism is one of the core concepts to understand the energetic functions of ATP synthase. Disulfide bonds formed by oxidizing a pair of cysteine mutations halted the rotation of the γ subunit in two critical conformations, the ATP-waiting dwell (αE284C/γQ274C) and the catalytic dwell (αE284C/γL276C). Tryptophan fluorescence was used to measure the nucleotide binding affinities for MgATP, MgADP and MgADP-AlF4 (a transition state analog) to wild-type and mutant F1 under reducing and oxidizing conditions. In the reduced state, αE284C/γL276C F1 showed a wild-type-like nucleotide binding pattern; after oxidation to lock the enzyme in the catalytic dwell state, the nucleotide binding parameters remained unchanged. In contrast, αE284C/γQ274C F1 showed significant differences in the affinities of the oxidized versus the reduced state. Locking the enzyme in the ATP-waiting dwell reduced nucleotide binding affinities of all three catalytic sites. Most importantly, the affinity of the low affinity site was reduced to such an extent that it could no longer be detected in the binding assay (Kd > 5 mM). The results of the present study allow to present a model for the catalytic mechanism of ATP synthase under consideration of the nucleotide affinity changes during a 360° cycle of the rotor.


Assuntos
Escherichia coli/enzimologia , Nucleotídeos/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Domínio Catalítico , Modelos Moleculares , Ligação Proteica
12.
J Chem Phys ; 154(16): 165103, 2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33940851

RESUMO

Studies of the effects of pressure on proteins from piezophilic (pressure-loving) microbes compared with homologous proteins from mesophilic microbes have been relatively rare. Interestingly, such studies of dihydrofolate reductase show that a single-site mutation from an aspartic acid to a glutamic acid can reverse the pressure-dependent monotonic decrease in activity to that in a monotonic pressure-dependent activation. This residue is near the active site but is not thought to directly participate in the catalytic mechanism. Here, the ways that addition of one carbon to the entire protein could lead to such a profound difference in pressure effects are explored using molecular dynamics simulations. The results indicate that the glutamate changes the coupling between a helix and the ß-sheet due to the extra flexibility of the side chain, which further changes correlated motions of other regions of the protein.


Assuntos
Pressão , Tetra-Hidrofolato Desidrogenase/química , Ácido Aspártico/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ácido Glutâmico/química , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Tetra-Hidrofolato Desidrogenase/genética
13.
Nat Commun ; 12(1): 2949, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011959

RESUMO

The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.


Assuntos
Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Resistência a Trimetoprima/genética , Trimetoprima/análogos & derivados , Substituição de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Cristalografia por Raios X , Evolução Molecular Direcionada , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Genes Bacterianos , Genótipo , Humanos , Modelos Moleculares , Mutação , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/química , Trimetoprima/farmacologia
14.
Int J Biol Macromol ; 182: 1473-1483, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34019922

RESUMO

κ-Carrageenan oligosaccharides with many excellent biological properties could be produced by κ-carrageenases selectively. In this study, based on the encoding gene of full length κ-carrageenase obtained from Pseudoalteromonas sp. ZDY3 and the reported mature secreted κ-carrageenase composed of 275 amino acid residues (N26-T300), CgkPZ_GH16 was expressed in E. coli, but no soluble active protein could be detected. Fortunately, the signal peptide of wild-type κ-carrageenase was recognized, and cleaved in the soluble and folding form in E. coli, the Km and kcat values of CgkPZ_SP_GH16 was 1.007 mg/mL and 362.8 s-1. By molecular dynamics simulations, it was showed that YjdB domain might affect the activity of κ-carrageenase. Due to the absence of mature processing modification system in E. coli, YjdB was remained in recombinant full length κ-carrageenase, and the lost catalytic efficiency of CgkPZ was compensated by expression level and thermal stability. Interestingly, CgkPZ_GH16_YjdB was expressed soluble without the signal peptide, which indicated that YjdB could contribute to the expression and folding of κ-carrageenase. These results provide new insight into the effects of different modules of κ-carrageenase on the expression and properties of enzyme.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/metabolismo , Pseudoalteromonas/enzimologia , Pseudoalteromonas/metabolismo , Proteínas de Bactérias/genética , Glicosídeo Hidrolases/genética
15.
Arch Microbiol ; 203(6): 3245-3255, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33837441

RESUMO

Drug users may represent a hidden reservoir of antibiotic resistance genes among their intestinal flora due to the poor hygiene and inappropriate use of antibiotics. Therefore, this study was focused to examine the prevalence of extended-spectrum ß-lactamase (ESBL) genes among intestinal Escherichia coli isolated from drug users in Ahvaz, Iran. Among clients of toxicology laboratory who were confirmed their addiction to each of Morphine, Amphetamine or Methamphetamine, 109 drug users were examined voluntarily for infection with hepatitis B or C using commercial enzyme linked immunosorbent assays (ELISA) method. Their stool specimens were obtained to isolate intestinal E. coli. The disc diffusion and combination disk methods were conducted to demonstrate antibiotic resistance pattern and phenotypically ESBL producers. ESBL-encoding genes (bla-TEM, bla-CTX-M, and bla-SHV) were also examined by PCR. Based on results, hepatitis C infection was more prevalent than hepatitis B among drug users. Of 109 isolates, a total of 57 (52.29%) ESBL positive E. coli were obtained from drug users and bla-TEM gene (60.55%) was found to be the most prevalent type, followed by bla-CTX-M (40.36%) and bla-SHV (39.44%). All isolates represented different resistance levels to tested antibiotics and 54.43% of the ESBL­producing isolates showed multidrug resistance (MDR) and the most frequent MDR pattern was simultaneous resistance to the seven (27.90%) of antimicrobials particularly erythromycin, penicillin, amoxycilin, cefteriaxon, cefotaxim, tetracycline and trimethoprim-Sulfamethoxazole. Fecal carriage of ESBL-production and MDR commensal isolates such as E. coli among drug users underlines the risk of transferring resistance genes between nonpathogenic and pathogenic bacteria.


Assuntos
Usuários de Drogas , Proteínas de Escherichia coli , Escherichia coli , Variação Genética , beta-Lactamases , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Humanos , Testes de Sensibilidade Microbiana , beta-Lactamases/genética
16.
J Biol Chem ; 296: 100699, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33895133

RESUMO

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alpha-anomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate.


Assuntos
Frutose-Bifosfato Aldolase/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Escherichia coli/enzimologia , Frutose-Bifosfato Aldolase/química , Modelos Moleculares , Ácido N-Acetilneuramínico/química , Periplasma/metabolismo , Conformação Proteica , Transporte Proteico , Estereoisomerismo
17.
Arch Biochem Biophys ; 704: 108874, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33862020

RESUMO

The short-chain flavin reductases BorF and AbeF reduce FAD to FADH2, which is then used by flavin-dependent halogenases (BorH and AbeH respectively) to regioselectively chlorinate tryptophan in the biosynthesis of indolotryptoline natural products. Recombinant AbeF and BorF were overexpressed and purified as homodimers from E. coli, and copurified with substoichiometric amounts of FAD, which could be easily removed. AbeF and BorF can reduce FAD, FMN, and riboflavin in vitro and are selective for NADH over NADPH. Initial velocity studies in the presence and absence of inhibitors showed that BorF proceeds by a sequential ordered kinetic mechanism in which FAD binds first, while AbeF follows a random-ordered sequence of substrate binding. Fluorescence quenching experiments verified that NADH does not bind BorF in the absence of FAD, and that both AbeF and BorF bind FAD with higher affinity than FADH2. pH-rate profiles of BorF and AbeF were bell-shaped with maximum kcat at pH 7.5, and site-directed mutagenesis of BorF implicated His160 and Arg38 as contributing to the catalytic activity and the pH dependence.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , FMN Redutase/química , Mononucleotídeo de Flavina/química , Flavina-Adenina Dinucleotídeo/química , Riboflavina/química , Cinética
18.
Nature ; 593(7857): 125-129, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33854236

RESUMO

Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis1-3. Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets4,5. The natural compound darobactin targets the bacterial insertase BamA6-the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins7-13. BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid ß-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Fenilpropionatos/química , Fenilpropionatos/farmacologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Desenho de Fármacos , Escherichia coli/citologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína
19.
Int J Mol Sci ; 22(7)2021 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-33801602

RESUMO

Supramolecular fibrous materials in biological systems play important structural and functional roles, and therefore, there is a growing interest in synthetic materials that mimic such fibrils, especially those bearing enzymatic reactivity. In this study, we investigated the self-assembly and enzymatic post-modification of short aromatic peptide amphiphiles (PAs), Fmoc-LnQG (n = 2 or 3), which contain an LQG recognition unit for microbial transglutaminase (MTG). These aromatic PAs self-assemble into fibrous structures via π-π stacking interactions between the Fmoc groups and hydrogen bonds between the peptides. The intermolecular interactions and morphologies of the assemblies were influenced by the solution pH because of the change in the ionization states of the C-terminal carboxy group of the peptides. Moreover, MTG-catalyzed post-modification of a small fluorescent molecule bearing an amine group also showed pH dependency, where the enzymatic reaction rate was increased at higher pH, which may be because of the higher nucleophilicity of the amine group and the electrostatic interaction between MTG and the self-assembled Fmoc-LnQG. Finally, the accumulation of the fluorescent molecule on these assembled materials was directly observed by confocal fluorescence images. Our study provides a method to accumulate functional molecules on supramolecular structures enzymatically with the morphology control.


Assuntos
Peptídeos/química , Transglutaminases/química , Aminas/química , Sítios de Ligação , Biomimética/métodos , Cadaverina/química , Ácidos Carboxílicos/química , Enzimas , Escherichia coli/enzimologia , Corantes Fluorescentes/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Microscopia Confocal , Nanoestruturas/química , Ligação Proteica , Domínios Proteicos , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática
20.
FEBS Lett ; 595(11): 1525-1541, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33792910

RESUMO

In the N-degron pathway of protein degradation of Escherichia coli, the N-recognin ClpS identifies substrates bearing N-terminal phenylalanine, tyrosine, tryptophan, or leucine and delivers them to the caseinolytic protease (Clp). Chloroplasts contain the Clp system, but whether chloroplastic ClpS1 adheres to the same constraints is unknown. Moreover, the structural underpinnings of substrate recognition are not completely defined. We show that ClpS1 recognizes canonical residues of the E. coli N-degron pathway. The residue in second position influences recognition (especially in N-terminal ends starting with leucine). N-terminal acetylation abrogates recognition. ClpF, a ClpS1-interacting partner, does not alter its specificity. Substrate binding provokes local remodeling of residues in the substrate-binding cavity of ClpS1. Our work strongly supports the existence of a chloroplastic N-degron pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Proteínas de Transporte/química , Cloroplastos/enzimologia , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cloroplastos/genética , Clonagem Molecular , Sequência Conservada , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Leucina/química , Leucina/metabolismo , Modelos Moleculares , Fenilalanina/química , Fenilalanina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Triptofano/química , Triptofano/metabolismo , Tirosina/química , Tirosina/metabolismo
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