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1.
Nature ; 609(7928): 808-814, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36104567

RESUMO

Complex I is the first enzyme in the respiratory chain, which is responsible for energy production in mitochondria and bacteria1. Complex I couples the transfer of two electrons from NADH to quinone and the translocation of four protons across the membrane2, but the coupling mechanism remains contentious. Here we present cryo-electron microscopy structures of Escherichia coli complex I (EcCI) in different redox states, including catalytic turnover. EcCI exists mostly in the open state, in which the quinone cavity is exposed to the cytosol, allowing access for water molecules, which enable quinone movements. Unlike the mammalian paralogues3, EcCI can convert to the closed state only during turnover, showing that closed and open states are genuine turnover intermediates. The open-to-closed transition results in the tightly engulfed quinone cavity being connected to the central axis of the membrane arm, a source of substrate protons. Consistently, the proportion of the closed state increases with increasing pH. We propose a detailed but straightforward and robust mechanism comprising a 'domino effect' series of proton transfers and electrostatic interactions: the forward wave ('dominoes stacking') primes the pump, and the reverse wave ('dominoes falling') results in the ejection of all pumped protons from the distal subunit NuoL. This mechanism explains why protons exit exclusively from the NuoL subunit and is supported by our mutagenesis data. We contend that this is a universal coupling mechanism of complex I and related enzymes.


Assuntos
Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons , Escherichia coli , Animais , Transporte de Elétrons , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli , Mutação , NAD/metabolismo , NADH Desidrogenase , Oxirredução , Subunidades Proteicas , Prótons , Quinonas/química , Quinonas/metabolismo , Eletricidade Estática , Água/química
2.
Proc Natl Acad Sci U S A ; 119(38): e2209608119, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36095194

RESUMO

Helicases are ubiquitous motor enzymes that remodel nucleic acids (NA) and NA-protein complexes in key cellular processes. To explore the functional repertoire and specificity landscape of helicases, we devised a screening scheme-Helicase-SELEX (Systematic Evolution of Ligands by EXponential enrichment)-that enzymatically probes substrate and cofactor requirements at global scale. Using the transcription termination Rho helicase of Escherichia coli as a prototype for Helicase-SELEX, we generated a genome-wide map of Rho utilization (Rut) sites. The map reveals many features, including promoter- and intrinsic terminator-associated Rut sites, bidirectional Rut tandems, and cofactor-dependent Rut sites with inverted G > C skewed compositions. We also implemented an H-SELEX variant where we used a model ligand, serotonin, to evolve synthetic Rut sites operating in vitro and in vivo in a ligand-dependent manner. Altogether, our data illustrate the power and flexibility of Helicase-SELEX to seek constitutive or conditional helicase substrates in natural or synthetic NA libraries for fundamental or synthetic biology discovery.


Assuntos
DNA Helicases , Riboswitch , Técnica de Seleção de Aptâmeros , Terminação da Transcrição Genética , Sítios de Ligação , DNA Helicases/química , Escherichia coli/enzimologia , Ligantes , Especificidade por Substrato
3.
Proc Natl Acad Sci U S A ; 119(37): e2123092119, 2022 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-36067314

RESUMO

Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. Escherichia coli encodes a dGTPase (2'-deoxyguanosine-5'-triphosphate [dGTP] triphosphohydrolase [dGTPase]; dgt gene, Dgt) that establishes the normal dGTP level required for accurate DNA replication but also plays a role in protecting E. coli against bacteriophage T7 infection by limiting the dGTP required for viral DNA replication. T7 counteracts Dgt using an inhibitor, the gene 1.2 product (Gp1.2). This interaction is a useful model system for studying the ongoing evolutionary virus/host "arms race." We determined the structure of Gp1.2 by NMR spectroscopy and solved high-resolution cryo-electron microscopy structures of the Dgt-Gp1.2 complex also including either dGTP substrate or GTP coinhibitor bound in the active site. These structures reveal the mechanism by which Gp1.2 inhibits Dgt and indicate that Gp1.2 preferentially binds the GTP-bound form of Dgt. Biochemical assays reveal that the two inhibitors use different modes of inhibition and bind to Dgt in combination to yield enhanced inhibition. We thus propose an in vivo inhibition model wherein the Dgt-Gp1.2 complex equilibrates with GTP to fully inactivate Dgt, limiting dGTP hydrolysis and preserving the dGTP pool for viral DNA replication.


Assuntos
Bacteriófago T7 , Proteínas de Escherichia coli , Escherichia coli , GTP Fosfo-Hidrolases , Guanosina Trifosfato , Proteínas Virais , Bacteriófago T7/fisiologia , Microscopia Crioeletrônica , Replicação do DNA , DNA Viral/metabolismo , Escherichia coli/enzimologia , Escherichia coli/virologia , Proteínas de Escherichia coli/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Conformação Proteica , Proteínas Virais/química , Replicação Viral
4.
Arch Microbiol ; 204(10): 627, 2022 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-36114886

RESUMO

Although Escherichia coli has four hydrogenases, their definite roles in fermentation are still not clear. In this study, all the operon deletion mutants of E.coli hydrogenases (∆hya, ∆hyb, ∆hyc, or ∆hyf) were constructed to evaluate the hydrogen metabolism in comparison to their respective single-gene deletion mutants of large subunits (∆hyaB, ∆hybC, ∆hycE, and ∆hyfG). Besides the hyc operon mutant that expectedly showed no hydrogen synthesis, the hyb operon mutant showed low hydrogen production and demonstrated significantly reduced growth under anaerobic conditions. The present work also provided first-hand data where deleterious effects of operon deletion were compared with single-gene deletion mutations and the results showed that the former type of deletion was found to cause more prominent phenotypic effects than the latter one. Interestingly, hyb operon mutant was remarkably distinct from other operon mutants, specifically in its inability to utilize glucose under both aerobic and anaerobic conditions. Further studies on this mutant revealed a significant reduction of the total intracellular ATP and NADH concentrations, which could explain its impaired glucose metabolism. In this way, Hyd-2 was verified as crucial not only in glucose metabolism but also in energy balance and redox homeostasis of the cells. Furthermore, a decreased expression of glucose metabolism-associated genes, particularly ppc and pykA, indicated their regulation by hyb operon, and thereby, glucose consumption. Moreover, the transcriptional changes in this mutant indicated the wide genomic connectivity of hyb operon to other metabolisms.


Assuntos
Escherichia coli/enzimologia , Hidrogenase , Trifosfato de Adenosina/metabolismo , Escherichia coli/genética , Glucose/metabolismo , Hidrogenase/genética , Hidrogenase/metabolismo , NAD/metabolismo , Óperon
5.
Nat Commun ; 13(1): 5502, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36127320

RESUMO

Enteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA-based pathway which is mobilised in response to aminoglycosides under anaerobiosis and engaged in cell membrane regulation.


Assuntos
Adenosina Trifosfatases , Aminoglicosídeos , Proteínas de Escherichia coli , Escherichia coli , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/metabolismo , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Fumaratos , Gentamicinas , Lipídeos de Membrana , Oxigênio/metabolismo , Fosfolipídeos
6.
Nature ; 609(7926): 384-393, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36002573

RESUMO

Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD1, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs2,3. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins4. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.


Assuntos
Adenosina Trifosfatases , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , RNA Bacteriano , Adenosina Trifosfatases/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Especificidade por Substrato , Transposases/metabolismo
7.
Proc Natl Acad Sci U S A ; 119(30): e2119368119, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35867824

RESUMO

Hypothiocyanite and hypothiocyanous acid (OSCN-/HOSCN) are pseudohypohalous acids released by the innate immune system which are capable of rapidly oxidizing sulfur-containing amino acids, causing significant protein aggregation and damage to invading bacteria. HOSCN is abundant in saliva and airway secretions and has long been considered a highly specific antimicrobial that is nearly harmless to mammalian cells. However, certain bacteria, commensal and pathogenic, are able to escape damage by HOSCN and other harmful antimicrobials during inflammation, which allows them to continue to grow and, in some cases, cause severe disease. The exact genes or mechanisms by which bacteria respond to HOSCN have not yet been elucidated. We have found, in Escherichia coli, that the flavoprotein RclA, previously implicated in reactive chlorine resistance, reduces HOSCN to thiocyanate with near-perfect catalytic efficiency and strongly protects E. coli against HOSCN toxicity. This is notable in E. coli because this species thrives in the chronically inflamed environment found in patients with inflammatory bowel disease and is able to compete with and outgrow other important commensal organisms, suggesting that HOSCN may be a relevant antimicrobial in the gut, which has not previously been explored. RclA is conserved in a variety of epithelium-colonizing bacteria, implicating its HOSCN reductase activity in a variety of host-microbe interactions. We show that an rclA mutant of the probiotic Limosilactobacillus reuteri is sensitive to HOSCN and that RclA homologs from Staphylococcus aureus, Streptococcus pneumoniae, and Bacteroides thetaiotaomicron all have potent protective activity against HOSCN when expressed in E. coli.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Oxirredutases , Tiocianatos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Tiocianatos/química , Tiocianatos/metabolismo
8.
Biophys J ; 121(16): 3103-3125, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35810330

RESUMO

Although it is relatively unexplored, accumulating data highlight the importance of tripartite crosstalk between nucleotide excision repair (NER), DNA replication, and recombination in the maintenance of genome stability; however, elucidating the underlying mechanisms remains challenging. While Escherichia coli uvrA and uvrB can fully complement polAΔ cells in DNA replication, uvrC attenuates this alternative DNA replication pathway, but the exact mechanism by which uvrC suppresses DNA replication is unknown. Furthermore, the identity of bona fide canonical and non-canonical substrates for UvrCs are undefined. Here, we reveal that Mycobacterium tuberculosis UvrC (MtUvrC) strongly binds to, and robustly cleaves, key intermediates of DNA replication/recombination as compared with the model NER substrates. Notably, inactivation of MtUvrC ATPase activity significantly attenuated its endonuclease activity, thus suggesting a causal link between these two functions. We built an in silico model of the interaction of MtUvrC with the Holliday junction (HJ), using a combination of homology modeling, molecular docking, and molecular dynamic simulations. The model predicted residues that were potentially involved in HJ binding. Six of these residues were mutated either singly or in pairs, and the resulting MtUvrC variants were purified and characterized. Among them, residues Glu595 and Arg597 in the helix-hairpin-helix motif were found to be crucial for the interaction between MtUvrC and HJ; consequently, mutations in these residues, or inhibition of ATP hydrolysis, strongly abrogated its DNA-binding and endonuclease activities. Viewed together, these findings expand the substrate specificity landscape of UvrCs and provide crucial mechanistic insights into the interplay between NER and DNA replication/recombination.


Assuntos
Endodesoxirribonucleases , Proteínas de Escherichia coli , Escherichia coli , Dano ao DNA , Reparo do DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Simulação de Acoplamento Molecular , Especificidade por Substrato
9.
Nucleic Acids Res ; 50(13): 7511-7528, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35819191

RESUMO

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor $\sigma$ to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein-DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RNAP-promoter open (RPO) complex is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting with $\sigma$ and the RNAP $\beta$ subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.


Assuntos
RNA Polimerases Dirigidas por DNA , Escherichia coli , Regiões Promotoras Genéticas , Bactérias/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Holoenzimas/genética , Holoenzimas/metabolismo , RNA Bacteriano , Fator sigma/metabolismo , Transcrição Genética
10.
Biochemistry ; 61(15): 1572-1584, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35861590

RESUMO

Glycosyltransferase (GT) enzymes promote the formation of glycosidic bonds between a sugar molecule and a diversity of substrates. Heptosyltransferase II (HepII) is a GT involved in the lipopolysaccharide (LPS) biosynthetic pathway that transfers the seven-carbon sugar (l-glycero-d-manno-heptose, Hep) onto a lipid-anchored glycopolymer (heptosylated Kdo2-Lipid A, Hep-Kdo2-Lipid A, or HLA). LPS plays a key role in Gram-negative bacterial sepsis, biofilm formation, and host colonization, and as such, LPS biosynthetic enzymes are targets for novel antimicrobial therapeutics. Three heptosyltransferases are involved in the inner-core LPS biosynthesis, with Escherichia coli HepII being the last to be quantitatively characterized in vivo. HepII shares modest sequence similarity with heptosyltransferase I (HepI) while maintaining a high degree of structural homology. Here, we report the first kinetic and biophysical characterization of HepII and demonstrate the properties of HepII that are shared with HepI, including sugar donor promiscuity and sugar acceptor-induced secondary structural changes, which results in significant thermal stabilization. HepII also has an increased catalytic efficiency and a significantly tighter binding affinity for both of its substrates compared to HepI. A structural model of the HepII ternary complex, refined by molecular dynamics simulations, was developed to probe the potentially important substrate-protein contacts. Ligand binding-induced changes in Trp fluorescence in HepII enabled the determination of substrate dissociation constants. Combined, these efforts meaningfully enhance our understanding of the heptosyltransferase family of enzymes and will aid in future efforts to design novel, potent, and specific inhibitors for this family of enzymes.


Assuntos
Escherichia coli , Glicosiltransferases , Lipídeo A , Catálise , Escherichia coli/enzimologia , Glicosiltransferases/metabolismo , Heptoses/química , Lipídeo A/metabolismo , Lipopolissacarídeos , Simulação de Dinâmica Molecular
11.
Commun Biol ; 5(1): 704, 2022 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-35835834

RESUMO

When overexpressed as an immature enzyme in the mesophilic bacterium Escherichia coli, recombinant homoserine dehydrogenase from the hyperthermophilic archaeon Sulfurisphaera tokodaii (StHSD) was markedly activated by heat treatment. Both the apo- and holo-forms of the immature enzyme were successively crystallized, and the two structures were determined. Comparison among the structures of the immature enzyme and previously reported structures of mature enzymes revealed that a conformational change in a flexible part (residues 160-190) of the enzyme, which encloses substrates within the substrate-binding pocket, is smaller in the immature enzyme. The immature enzyme, but not the mature enzyme, formed a complex that included NADP+, despite its absence during crystallization. This indicates that the opening to the substrate-binding pocket in the immature enzyme is not sufficient for substrate-binding, efficient catalytic turnover or release of NADP+. Thus, specific conformational changes within the catalytic region appear to be responsible for heat-induced activation.


Assuntos
Escherichia coli/enzimologia , Homosserina Desidrogenase/química , Homosserina Desidrogenase/metabolismo , Temperatura Alta , Sulfolobaceae/enzimologia , Domínio Catalítico/fisiologia , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , NADP/química , NADP/metabolismo
12.
Chembiochem ; 23(15): e202200293, 2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35648642

RESUMO

Temperature is a crucial parameter for biological and chemical processes. Its effect on enzymatically catalysed reactions has been known for decades, and stereo- and enantiopreference are often temperature-dependent. For the first time, we present the temperature effect on the Baeyer-Villiger oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one by the type II Bayer-Villiger monooxygenase, 2,5-DKCMO. In the absence of a reductase and driven by the hydride-donation of a synthetic nicotinamide analogue, the clear trend for a decreasing enantioselectivity at higher temperatures was observed. "Traditional" approaches such as the determination of the enantiomeric ratio (E) appeared unsuitable due to the complexity of the system. To quantify the trend, we chose to use the 'Shape Language Modelling' (SLM), a tool that allows the reaction to be described at all points in a shape prescriptive manner. Thus, without knowing the equation of the reaction, the substrate ee can be estimated that at any conversion.


Assuntos
Escherichia coli , Oxigenases de Função Mista , Escherichia coli/enzimologia , Oxigenases de Função Mista/metabolismo , Oxirredução , Temperatura
13.
J Biol Chem ; 298(8): 102203, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35764173

RESUMO

Lipoic acid is a sulfur-containing cofactor indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential actions of lipoate protein ligase B (LipB) and lipoyl synthase (LipA). LipB takes up the octanoyl chain from C8-acyl carrier protein (C8-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. However, the molecular basis of its substrate recognition is still not fully understood. Using Escherichia coli LipB as a model enzyme, we show here that the octanoyl-transferase mainly recognizes the 4'-phosphopantetheine-tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. We demonstrate LipB can accept octanoate from its own ACP and noncognate ACPs, as well as C8-CoA. Furthermore, our 1H saturation transfer difference and 31P NMR studies demonstrate the binding of adenosine, as well as the phosphopantetheine arm of CoA to LipB, akin to binding to LplA. Finally, we show a conserved 71RGG73 loop, analogous to the lipoate-binding loop of LplA, is required for full LipB activity. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge could be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.


Assuntos
Aciltransferases/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteína de Transporte de Acila/metabolismo , Aciltransferases/metabolismo , Coenzima A/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligases/metabolismo , Panteteína/análogos & derivados , Ácido Tióctico/metabolismo
14.
Annu Rev Microbiol ; 76: 533-552, 2022 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-35671533

RESUMO

RNA degradosomes are multienzyme complexes composed of ribonucleases, RNA helicases, and metabolic enzymes. RNase E-based degradosomes are widespread in Proteobacteria. The Escherichia coli RNA degradosome is sequestered from transcription in the nucleoid and translation in the cytoplasm by localization to the inner cytoplasmic membrane, where it forms short-lived clusters that are proposed to be sites of mRNA degradation. In Caulobacter crescentus, RNA degradosomes localize to ribonucleoprotein condensates in the interior of the cell [bacterial ribonucleoprotein-bodies (BR-bodies)], which have been proposed to drive the concerted degradation of mRNA to nucleotides. The turnover of mRNA in growing cells is important for maintaining pools of nucleotides for transcription and DNA replication.Membrane attachment of the E. coli RNA degradosome is necessary to avoid wasteful degradation of intermediates in ribosome assembly. Sequestering RNA degradosomes to C. crescentus BR-bodies, which exclude structured RNA, could have a similar role in protecting intermediates in ribosome assembly from degradation.


Assuntos
Caulobacter crescentus , Endorribonucleases , Escherichia coli , Complexos Multienzimáticos , Nucleotídeos , Polirribonucleotídeo Nucleotidiltransferase , RNA Helicases , Estabilidade de RNA , RNA Mensageiro , Caulobacter crescentus/enzimologia , Caulobacter crescentus/genética , Endorribonucleases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Nucleotídeos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo
15.
Infect Dis Now ; 52(6): 334-340, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35700962

RESUMO

OBJECTIVES: The emergence and spread of Carbapenem-Resistant Enterobacterales (CRE) has become a growing concern for health services, internationally, nationally, and regionally. In Morocco, the situation is more worrisome as studies on CRE are scarce and/or scattered and/or outdated. As a result, we carried out the present study to determine and update CRE prevalence at Mohammed VI University Hospital of Marrakech, Morocco. PATIENTS AND METHODS: A cross-sectional prospective study was carried out from March 2018 to March 2020 on 41161 clinical specimens of 23,469 patients suspected of bacterial infections. Enterobacterales strains were isolated following standard bacteriological procedures. Bacterial strains were identified using BD-Phoenix and MALDI-TOF-MS. Antibiotic susceptibility was determined for 14 antibiotics. Carbapenemase production and phenotypic detection were characterized using modified carbapenem inactivation phenotypic and immunochromatographic methods. RESULTS: All in all, 484 Enterobaterales resistant to at least one carbapenem were recovered. The majority was isolated from the neonatal unit (14%), followed by the urology-nephrology (11%), and plastic surgery departments (10%). K. pneumoniae (n=232) was the most isolated, followed by E. cloacae (n=148), E. coli (n=56), and S. marcescens (n=17). Antibiotic susceptibility profile showed high rates of resistance to ciprofloxacin (75.21%), gentamicin (84.50%), and cotrimoxazole (88.42%). Out of 484 CRE positive cultures, 388 (80.16%) were Carbapenemase-positive. Out of the latter, 170 were metallo-beta-lactamase producers (NDM), 162 OXA-48-like, and 56 both. CONCLUSION: These findings emphasize the urgent need for control precautions and strict measures to contain and mitigate this issue.


Assuntos
Antibacterianos , Proteínas de Bactérias , Enterobacteriáceas Resistentes a Carbapenêmicos , Carbapenêmicos , beta-Lactamases , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Carbapenêmicos/farmacologia , Estudos Transversais , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Hospitais , Hospitais Universitários , Humanos , Recém-Nascido , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Marrocos/epidemiologia , Estudos Prospectivos , beta-Lactamases/metabolismo
16.
J Biol Chem ; 298(7): 102099, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35667439

RESUMO

Bacterial RNA polymerase (RNAP) coordinates transcription with DNA repair and replication. Many RNAP mutations have pleiotropic phenotypes with profound effects on transcription-coupled processes. One class of RNAP mutations (rpo∗) has been shown to suppress mutations in regulatory factors responsible for changes in gene expression during stationary phase or starvation, as well as in factors involved in the restoration of replication forks after DNA damage. These mutations were suggested to affect the ability of RNAP to transcribe damaged DNA and to decrease the stability of transcription complexes, thus facilitating their dislodging during DNA replication and repair, although this was not explicitly demonstrated. Here, we obtained nine mutations of this class located around the DNA/RNA binding cleft of Escherichia coli RNAP and analyzed their transcription properties in vitro. We found that these mutations decreased promoter complex stability to varying degrees, and all decreased the activity of rRNA promoters. However, they did not have strong effects on elongation complex stability. Some mutations were shown to stimulate transcriptional pauses or decrease intrinsic RNA cleavage by RNAP, but none altered the ability of RNAP to transcribe DNA templates containing damaged nucleotides. Thus, we conclude that the suppressor phenotypes of the mutations are unlikely to result from direct effects on DNA lesion recognition by RNAP but may be primarily explained by changes in transcription initiation. Further analysis of the effects of these mutations on the genomic distribution of RNAP and its interactions with regulatory factors will be essential for understanding their diverse phenotypes in vivo.


Assuntos
RNA Polimerases Dirigidas por DNA , Proteínas de Escherichia coli , Escherichia coli , Supressão Genética , Reparo do DNA , Replicação do DNA , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , Transcrição Genética
17.
Proc Natl Acad Sci U S A ; 119(25): e2202022119, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35714287

RESUMO

The enzyme ribonucleotide reductase (RNR), which catalyzes the reduction of ribonucleotides to deoxynucleotides, is vital for DNA synthesis, replication, and repair in all living organisms. Its mechanism requires long-range radical translocation over ∼32 Šthrough two protein subunits and the intervening aqueous interface. Herein, a kinetic model is designed to describe reversible radical transfer in Escherichia coli RNR. This model is based on experimentally studied photoRNR systems that allow the photochemical injection of a radical at a specific tyrosine residue, Y356, using a photosensitizer. The radical then transfers across the interface to another tyrosine residue, Y731, and continues until it reaches a cysteine residue, C439, which is primed for catalysis. This kinetic model includes radical injection, an off-pathway sink, radical transfer between pairs of residues along the pathway, and the conformational flipping motion of Y731 at the interface. Most of the input rate constants for this kinetic model are obtained from previous experimental measurements and quantum mechanical/molecular mechanical free-energy simulations. Ranges for the rate constants corresponding to radical transfer across the interface are determined by fitting to the experimentally measured Y356 radical decay times in photoRNR systems. This kinetic model illuminates the time evolution of radical transport along the tyrosine and cysteine residues following radical injection. Further analysis identifies the individual rate constants that may be tuned to alter the timescale and probability of the injected radical reaching C439. The insights gained from this kinetic model are relevant to biochemical understanding and protein-engineering efforts with potential pharmacological implications.


Assuntos
Cisteína , Proteínas de Escherichia coli , Escherichia coli , Ribonucleotídeo Redutases , Cisteína/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Modelos Químicos , Simulação de Dinâmica Molecular , Ribonucleotídeo Redutases/química , Termodinâmica , Tirosina/química
18.
Protein Expr Purif ; 197: 106112, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35598696

RESUMO

CCP6 is a member of cytosolic carboxypeptidases (CCPs) family, an eraser of a reversible protein posttranslational modification - polyglutamylation, and represents a potential therapeutic target. Currently, production of CCPs mainly depends on eukaryotic expression system, which is time-consuming and costly. Here, we reported that mouse origin full-length CCP6 can be successfully expressed in the soluble fraction of bacteria ArcticExpress (DE3) strain. However, the recombinant mCCP6 was initially co-purified with Cpn60 in a stoichiometric ratio of roughly 1:7 and exhibited no enzyme activity. When coupled with a step to promote the release of the substrate protein from the chaperonins by treatment with ATP/Mg2+/K+, the recombinant CCP6 with deglutamylation activity was obtained, though still partially associated with Cpn60. This is the first report, to our knowledge, that the successful expression and purification of active recombinant mammalian CCPs using a bacterial system was achieved.


Assuntos
Carboxipeptidases , Escherichia coli , Animais , Carboxipeptidases/genética , Carboxipeptidases/isolamento & purificação , Carboxipeptidases/metabolismo , Chaperonina 60/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Mamíferos , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
19.
Bioorg Chem ; 122: 105739, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35306417

RESUMO

Bacterial tRNA 2-selenouridine synthase (SelU) in vitro converts S2U-RNA to its selenium analog (Se2U-RNA) in a two-step process: (i) geranylation of S2U-RNA (with geranyl pyrophosphate, gePP), and (ii) selenation of the resulting geS2U-RNA (with the selenophosphate anion, SePO33-). Using an S2U-containing anticodon stem-loop fragment derived from tRNALys (S2U-RNA) and recombinant SelU with an MBP tag, we found that only geranyl (C10) pyrophosphate is the substrate for this enzyme, while other pyrophosphates such as isopentenyl (C5), dimethylallyl (C5), farnesyl (C15) and geranylgeranyl (C20) are not. Interestingly, methyl (C1)- and C5-, C10-, and C15-prenyl-containing S2U-RNAs (which were chemically obtained) underwent the selenation reaction promoted by SelU, although the Se2U-RNA product was obtained in decreasing yields in the following order: geranyl ≥ farnesyl > dimethylallyl ≫ methyl. Microscale thermophoresis showed an affinity between gePP and SelU in the micromolar range, while the other pyrophosphates tested, such as isopentenyl, dimethylallyl, farnesyl and geranylgeranyl, either did not bind to the protein or their binding affinity was above 1 mM. These results agree well with the in silico analysis, with gePP being the best binding substrate (the lowest relative free energy of binding (ΔG) and a small solvent-accessible surface area (SASA)). These results suggest that SelU has high substrate specificity for the prenylation reaction (only gePP is accepted), whereas there is little discrimination for the selenation reaction. We therefore suggest that only gePP and the geranylated tRNA serve as substrates for the conversion of 2-thio-tRNAs to 2-seleno-tRNAs, as it is found in the bacterial system.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Selênio , Sulfurtransferases , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Neopreno , Sulfurtransferases/genética , Sulfurtransferases/metabolismo
20.
Biochemistry ; 61(7): 608-615, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35255690

RESUMO

Carrier protein-dependent biosynthesis provides a thiotemplated format for the production of natural products. Within these pathways, many reactions display exquisite substrate selectivity, a regulatory framework proposed to be controlled by protein-protein interactions (PPIs). In Escherichia coli, unsaturated fatty acids are generated within the de novo fatty acid synthase by a chain length-specific interaction between the acyl carrier protein AcpP and the isomerizing dehydratase FabA. To evaluate PPI-based control of reactivity, interactions of FabA with AcpP bearing multiple sequestered substrates were analyzed through NMR titration and guided high-resolution docking. Through a combination of quantitative binding constants, residue-specific perturbation analysis, and high-resolution docking, a model for substrate control via PPIs has been developed. The in silico results illuminate the mechanism of FabA substrate selectivity and provide a structural rationale with atomic detail. Helix III positioning in AcpP communicates sequestered chain length identity recognized by FabA, demonstrating a powerful strategy to regulate activity by allosteric control. These studies broadly illuminate carrier protein-dependent pathways and offer an important consideration for future inhibitor design and pathway engineering.


Assuntos
Proteína de Transporte de Acila , Ácido Graxo Sintase Tipo II , Ácidos Graxos , Hidroliases , Proteína de Transporte de Acila/metabolismo , Escherichia coli/enzimologia , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos Insaturados/metabolismo , Hidroliases/metabolismo
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