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1.
Microbiol Res ; 223-225: 88-98, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178056

RESUMO

CodY and (p)ppGpp synthetases are two important global regulators of bacteria. In some pathogens, such as Listeria monocytogenes, the GTP pool links these two regulatory systems, and introducing a codY mutant into the ΔrelA strain restored the pathogenicity of the attenuated ΔrelA mutant. In previous studies, we identified the (p)ppGpp synthetases (RelA and RelQ) and CodY of Streptococcus suis. To understand the interrelationships between these two regulators in S. suis, a ΔrelAΔrelQΔcodY mutant was constructed, and its growth, morphology, and pathogenicity were evaluated. Compared with ΔrelAΔrelQ, ΔcodY, its growth was very slow, but its chain length was partly restored to the wild-type length and its capsule became thick and rough. The adherence, invasion ability, and resistance to whole-blood killing in vitro of ΔrelAΔrelQΔcodY and its lethality and colonization ability in mice were clearly reduced, which differs from the effects of these mutations in L. monocytogenes. An analysis of gene expression showed that CodY interacted with the relA promoter in a GTP-independent manner to positively regulate the expression of relA. The introduction of a codY mutant into the ΔrelAΔrelQ strain further reduced the expression of virulence factors, which suggests a novel interaction between the (p)ppGpp synthetases and CodY. This study extends our understanding of the relationship between the (p)ppGpp-mediated stringent response and the regulation of CodY in S. suis.


Assuntos
Regulação Bacteriana da Expressão Gênica , Ligases/metabolismo , Streptococcus suis/citologia , Streptococcus suis/metabolismo , Streptococcus suis/patogenicidade , Fatores de Transcrição/metabolismo , Transcriptoma , Adesinas Bacterianas/genética , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Guanosina Trifosfato/metabolismo , Ligases/genética , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Camundongos , Mutação , Regiões Promotoras Genéticas , Infecções Estreptocócicas/microbiologia , Streptococcus suis/genética , Fatores de Transcrição/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
2.
Plant Physiol Biochem ; 141: 206-214, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31176880

RESUMO

SUMOylation is an important protein modification that regulates the properties of substrate proteins in a variety of cellular processes. SUMOylation is catalyzed via a cascade of enzymes and is usually stimulated by SUMO E3 ligases. However, the molecular functions and regulatory mechanisms of SUMOylation in forage crops are unknown. Here, we isolated and functionally characterized DiMMS21, a homolog of the Arabidopsis thaliana SUMO ligase AtMMS21, from the forage legume Desmodium intortum. DiMMS21 is expressed ubiquitously in various D. intortum organs and its encoded protein is found in the cytoplasm and nucleus. Bioinformatics analysis indicated that DiMMS21 contains a conserved SP-RING domain that is required for its activity. Biochemical evidence supports the notion that this protein is a functional SUMO ligase. When expressed in an Arabidopsis mms21 mutant, DiMMS21 completely rescued the defects in root, leaf, and silique development. The results from cotyledon greening and marker gene expression suggested that DiMMS21 can only partially complements the role of AtMMS21 in abscisic acid (ABA) responses. In summary, we characterized the molecular features of DiMMS21 and uncovered potential roles of this SUMO ligase in development and ABA responses, increasing our understanding on the function of SUMOylation in forage crops.


Assuntos
Fabaceae/enzimologia , Regulação da Expressão Gênica de Plantas , Ligases/metabolismo , Proteínas de Plantas/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Biologia Computacional , Citoplasma/metabolismo , DNA Complementar/metabolismo , Perfilação da Expressão Gênica , Mutação , Filogenia , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Sementes/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
3.
J Enzyme Inhib Med Chem ; 34(1): 1010-1017, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31072165

RESUMO

The Mur ligases form a series of consecutive enzymes that participate in the intracellular steps of bacterial peptidoglycan biosynthesis. They therefore represent interesting targets for antibacterial drug discovery. MurC, D, E and F are all ATP-dependent ligases. Accordingly, with the aim being to find multiple inhibitors of these enzymes, we screened a collection of ATP-competitive kinase inhibitors, on Escherichia coli MurC, D and F, and identified five promising scaffolds that inhibited at least two of these ligases. Compounds 1, 2, 4 and 5 are multiple inhibitors of the whole MurC to MurF cascade that act in the micromolar range (IC50, 32-368 µM). NMR-assisted binding studies and steady-state kinetics studies performed on aza-stilbene derivative 1 showed, surprisingly, that it acts as a competitive inhibitor of MurD activity towards D-glutamic acid, and additionally, that its binding to the D-glutamic acid binding site is independent of the enzyme closure promoted by ATP.


Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Ligases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Cinética , Ligases/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade
4.
FEMS Microbiol Rev ; 43(4): 389-400, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980074

RESUMO

Bacteria use dedicated mechanisms to respond adequately to fluctuating environments and to optimize their chances of survival in harsh conditions. One of the major stress responses used by virtually all bacteria relies on the sharp accumulation of an alarmone, the guanosine penta- or tetra-phosphate commonly referred to as (p)ppGpp. Under stressful conditions, essentially nutrient starvation, these second messengers completely reshape the metabolism and physiology by coordinately modulating growth, transcription, translation and cell cycle. As a central regulator of bacterial stress response, the alarmone is also involved in biofilm formation, virulence, antibiotics tolerance and resistance in many pathogenic bacteria. Intracellular concentrations of (p)ppGpp are determined by a highly conserved and widely distributed family of proteins called RelA-SpoT Homologs (RSH). Recently, several studies uncovering mechanisms that regulate RSH activities have renewed a strong interest in this field. In this review, we outline the diversity of the RSH protein family as well as the molecular devices used by bacteria to integrate and transform environmental cues into intracellular (p)ppGpp levels.


Assuntos
Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Hidrolases/genética , Ligases/genética , Proteínas de Bactérias/metabolismo , Hidrolases/metabolismo , Ligases/metabolismo , Estresse Fisiológico/genética , Virulência/genética
5.
Chemosphere ; 225: 608-617, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30901654

RESUMO

Acyl-homoserine lactone (AHL)-based quorum sensing (QS) in the anaerobic ammonium oxidizing (anammox) consortia has attracted increasing attention. However, AHL synthase in anammox bacteria and the relationship between AHL synthetic genes and anammox activity are still not clear because anammox bacteria have not been isolated from the consortia. Two novel synthases of AHLs (JqsI-1 and JqsI-2), which are HdtS-type rather than the widely studied LuxI-type, were identified in anammox bacteria Candidatus Jettenia caeni and synthesized four AHLs. There was a correlation between AHL concentration, in situ transcriptional expression of the AHL synthase genes (jqsI-1 and jqsI-2) and genetic marker of anammox activity (hydrazine synthase gene, hzsA). And AHL add-back studies demonstrated that AHL influence the expression of hzsA to regulate anammox bacterial activity. This study provides insight into the QS communication pathway of anammox bacteria for wastewater treatment.


Assuntos
Acil-Butirolactonas/metabolismo , Bactérias/enzimologia , Ligases/metabolismo , Consórcios Microbianos , Percepção de Quorum , Aciltransferases , Compostos de Amônio/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias , Biodegradação Ambiental , Ligases/genética , Fatores de Transcrição , Transcrição Genética , Águas Residuárias/microbiologia
6.
Plant Sci ; 280: 314-320, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30824010

RESUMO

The 26S proteasome is a multi-subunit protease controlling most of the cytosolic and nuclear protein turnover, regulating many cellular events in eukaryotes. However, functional modification on this complex remains unclear. Here, we showed a novel mechanism that a SUMO ligase AtMMS21 regulates activity of the 26S proteasome in root development of Arabidopsis. Our in vitro and in vivo data supported that AtMMS21 interacts with RPT2a, a subunit of the 26S proteasome. The mutants of AtMMS21 and RPT2a display similar developmental defect of roots, suggesting their association in this process. In addition, RPT2a is modified by SUMO3, potentially related to AtMMS21. During development, the activity of the 26S proteasome is lower in both mutants of AtMMS21 and RPT2a, compared with that of wild type. Furthermore, the protein level but not the RNA level of RPT2a is decreased in the absence of AtMMS21, implying stability regulation of the proteasome complex through the AtMMS21-RPT2a interaction. Taken together, the current study would improve our understanding on the regulatory mechanism of the 26S proteasome via protein modification in root development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Ligases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Ligases/genética , Mutação , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Complexo de Endopeptidases do Proteassoma/genética , Sumoilação
7.
Microbiol Res ; 220: 32-41, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30744817

RESUMO

The type VI secretion system (T6SS) is a versatile molecular machinery widely distributed in Gram-negative bacteria. The activity of the T6SS is tightly regulated by various mechanisms, including quorum sensing (QS), iron concentration, and transcriptional regulators. Here we demonstrated that the stringent response regulator, RelA, contributes to bacterial resistance to multiple environmental stresses in Yersinia pseudotuberculosis. We also revealed that the stress resistance function of stringent response (SR) was partially mediated by the general stress response T6SS4 system. RelA positively regulates the expression of T6SS4 to combat various stresses in response to nutrition starvation collectively mediated by the RovM and RovA regulators. These findings revealed not only the important role of T6SS4 in SR induced stress resistance, but also a new pathway to regulate T6SS4 expression in response to starvation stress.


Assuntos
Proteínas de Bactérias/metabolismo , GTP Pirofosfoquinase/metabolismo , Ligases/metabolismo , Fatores de Transcrição/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/genética , Ligases/genética , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas/genética , Inanição , Estresse Fisiológico , Sistemas de Secreção Tipo VI/genética , Yersinia pseudotuberculosis/genética
8.
PLoS Genet ; 15(2): e1007942, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30735491

RESUMO

NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2.


Assuntos
Dano ao DNA , Ligases/metabolismo , Mitose , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Epistasia Genética , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Hidroxiureia/farmacologia , Ligases/deficiência , Ligases/genética , Modelos Biológicos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , RecQ Helicases/deficiência , RecQ Helicases/genética , RecQ Helicases/metabolismo , Troca de Cromátide Irmã/efeitos dos fármacos , Sumoilação
9.
Org Lett ; 21(7): 2029-2032, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30763108

RESUMO

A biomimetic one-step ligase-catalyzed cyclo-oligomerization mediated by butelase 1, an Asn/Asp-specific ligase, is introduced that is time-, concentration-, length-, and sequence-dependent. This reaction yields cyclic mono-, di-, tri-, and tetramers from peptide precursors containing 3-15 amino acids ended with Asn and a His-Val tail. The cyclomonomers were favored when the peptide lengths were >9 amino acids. A turn-forming Pro residue at the P2 position favored the formation of higher-order cyclo-oligomers.


Assuntos
Aminoácidos/química , Depsipeptídeos/química , Ligases/química , Peptídeos Cíclicos/química , Aminoácidos/metabolismo , Catálise , Depsipeptídeos/metabolismo , Ligases/metabolismo , Estrutura Molecular , Peptídeos Cíclicos/metabolismo
10.
Nat Plants ; 5(2): 225-237, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30692678

RESUMO

Lignin is the main cause of lignocellulosic biomass recalcitrance to industrial enzymatic hydrolysis. By partially replacing the traditional lignin monomers by alternative ones, lignin extractability can be enhanced. To design a lignin that is easier to degrade under alkaline conditions, curcumin (diferuloylmethane) was produced in the model plant Arabidopsis thaliana via simultaneous expression of the turmeric (Curcuma longa) genes DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE 2 (CURS2). The transgenic plants produced a plethora of curcumin- and phenylpentanoid-derived compounds with no negative impact on growth. Catalytic hydrogenolysis gave evidence that both curcumin and phenylpentanoids were incorporated into the lignifying cell wall, thereby significantly increasing saccharification efficiency after alkaline pretreatment of the transgenic lines by 14-24% as compared with the wild type. These results demonstrate that non-native monomers can be synthesized and incorporated into the lignin polymer in plants to enhance their biomass processing efficiency.


Assuntos
Arabidopsis/metabolismo , Curcumina/metabolismo , Lignina/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Arabidopsis/genética , Biomassa , Parede Celular/genética , Parede Celular/metabolismo , Celulose/metabolismo , Curcuma/genética , Glucose/metabolismo , Ligases/genética , Ligases/metabolismo , Lignina/genética , Proteínas de Plantas/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Temperatura Ambiente
11.
J Agric Food Chem ; 67(5): 1453-1462, 2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30638374

RESUMO

ε-Poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide, which is generally recognized as safe for use as a food preservative. To date, the production capacity of strains that produce low-molecular weight ε-PL remains very low and thus unsuitable for industrial production. Here, we report a new low-molecular weight ε-PL-producing Kitasatospora aureofaciens strain. The ε-PL synthase gene of this strain was cloned into a high ε-PL-producing Streptomyces albulus strain. The resulting recombinant strain efficiently produced ε-PL with a molecular weight of 1.3-2.3 kDa and yielded of 23.6 g/L following fed-batch fermentation in a 5 L bioreactor. In addition, circular dichroism spectra showed that this ε-PL takes on a conformation similar to an antiparallel pleated-sheet. Moreover, it demonstrated better antimicrobial activity against yeast compared to the 3.2-4.5 kDa ε-PL. This study provides a highly efficient strategy for production of the low-molecular weight ε-PL, which helps to expand its potential applications.


Assuntos
Proteínas de Bactérias/genética , Ligases/genética , Polilisina/biossíntese , Streptomyces/metabolismo , Streptomycetaceae/enzimologia , Antifúngicos/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Fermentação , Ligases/metabolismo , Polilisina/química , Polilisina/farmacologia , Streptomyces/genética , Streptomycetaceae/genética , Leveduras/efeitos dos fármacos
12.
Curr Genet ; 65(3): 669-676, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30600397

RESUMO

Duplication of the genome poses one of the most significant threats to genetic integrity, cellular fitness, and organismal health. Therefore, numerous mechanisms have evolved that maintain replication fork stability in the face of DNA damage and allow faithful genome duplication. The fission yeast BRCT-domain-containing protein Brc1, and its budding yeast orthologue Rtt107, has emerged as a "hub" factor that integrates multiple replication fork protection mechanisms. Notably, the cofactors and pathways through which Brc1, Rtt107, and their human orthologue (PTIP) act have appeared largely distinct. This either represents true evolutionary functional divergence, or perhaps an incomplete genetic and biochemical analysis of each protein. In this regard, we recently showed that like Rtt107, Brc1 supports key functions of the Smc5-Smc6 complex, including its recruitment into DNA repair foci, chromatin association, and SUMO ligase activity. Furthermore, fission yeast cells lacking the Nse5-Nse6 genome stability factor were found to exhibit defects in Smc5-Smc6 function, similar to but more severe than those in cells lacking Brc1. Here, we place these findings in context with the known functions of Brc1, Rtt107, and Smc5-Smc6, present data suggesting a role for acetylation in Smc5-Smc6 chromatin loading, and discuss wider implications for genome stability.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Instabilidade Genômica , Ligases/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Dano ao DNA , Replicação do DNA , Ligases/genética , Proteína SUMO-1/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
13.
PLoS Pathog ; 15(1): e1007543, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30689667

RESUMO

Merkel cell polyomavirus (MCPyV) accounts for 80% of all Merkel cell carcinoma (MCC) cases through expression of two viral oncoproteins: the truncated large T antigen (LT-t) and small T antigen (ST). MCPyV ST is thought to be the main driver of cellular transformation and has also been shown to increase LT protein levels through the activity of its Large-T Stabilization Domain (LSD). The ST LSD was reported to bind and sequester several ubiquitin ligases, including Fbw7 and ß-TrCP, and thereby stabilize LT-t and several other Fbw7 targets including c-Myc and cyclin E. Therefore, the ST LSD is thought to contribute to transformation by promoting the accumulation of these oncoproteins. Targets of Fbw7 and ß-TrCP contain well-defined, conserved, phospho-degrons. However, as neither MCPyV LT, LT-t nor ST contain the canonical Fbw7 phospho-degron, we sought to further investigate the proposed model of ST stabilization of LT-t and transformation. In this study, we provide several lines of evidence that fail to support a specific interaction between MCPyV T antigens and Fbw7 or ß-TrCP by co-immunoprecipitation or functional consequence. Although MCPyV ST does indeed increase LT protein levels through its Large-T Stabilization domain (LSD), this is accomplished independently of Fbw7. Therefore, our study indicates a need for further investigation into the role and mechanism(s) of MCPyV T antigens in viral replication, latency, transformation, and tumorigenesis.


Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Proteína 7 com Repetições F-Box-WD/metabolismo , Poliomavírus das Células de Merkel/metabolismo , Antígenos de Neoplasias/metabolismo , Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/metabolismo , Células HEK293 , Humanos , Ligases/metabolismo , Células de Merkel , Poliomavírus das Células de Merkel/imunologia , Poliomavírus das Células de Merkel/patogenicidade , Proteínas Oncogênicas/metabolismo , Infecções por Polyomavirus/metabolismo , Domínios Proteicos , Infecções Tumorais por Vírus/virologia , Ubiquitina/metabolismo , Replicação Viral , Proteínas Contendo Repetições de beta-Transducina/metabolismo
14.
Org Biomol Chem ; 17(2): 234-239, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30556075

RESUMO

Use of the ku70-deficient strain of Coprinopsis cinerea enabled confirmation within the native context of the central role the sesquiterpene synthase Cop6 plays in lagopodin biosynthesis. Furthermore, yeast in vivo bioconversion and in vitro assays of two cytochrome P450 monooxygenases Cox1 and Cox2 allowed elucidation of the network of oxidation steps that build structural complexity onto the α-cuprenene framework during the biosynthesis of lagopodins. Three new compounds were identified as intermediates formed by the redox enzymes.


Assuntos
Coprinus/enzimologia , Coprinus/metabolismo , Sesquiterpenos/metabolismo , Vias Biossintéticas , Coprinus/química , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas Fúngicas/metabolismo , Ligases/metabolismo , Oxirredução , Quinonas/química , Quinonas/metabolismo , Sesquiterpenos/química
15.
Biochemistry ; 58(6): 833-847, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30582694

RESUMO

There is a paramount need for expanding the drug armamentarium to counter the growing problem of drug-resistant tuberculosis. Salicyl-AMS, an inhibitor of salicylic acid adenylation enzymes, is a first-in-class antibacterial lead compound for the development of tuberculosis drugs targeting the biosynthesis of salicylic-acid-derived siderophores. In this study, we determined the Ki of salicyl-AMS for inhibition of the salicylic acid adenylation enzyme MbtA from Mycobacterium tuberculosis (MbtAtb), designed and synthesized two new salicyl-AMS analogues to probe structure-activity relationships (SAR), and characterized these two analogues alongside salicyl-AMS and six previously reported analogues in biochemical and cell-based studies. The biochemical studies included determination of kinetic parameters ( Kiapp, konapp, koff, and tR) and analysis of the mechanism of inhibition. For these studies, we optimized production and purification of recombinant MbtAtb, for which Km and kcat values were determined, and used the enzyme in conjunction with an MbtAtb-optimized, continuous, spectrophotometric assay for MbtA activity and inhibition. The cell-based studies provided an assessment of the antimycobacterial activity and postantibiotic effect of the nine MbtAtb inhibitors. The antimycobacterial properties were evaluated using a strain of nonpathogenic, fast-growing Mycobacterium smegmatis that was genetically engineered for MbtAtb-dependent susceptibility to MbtA inhibitors. This convenient model system greatly facilitated the cell-based studies by bypassing the methodological complexities associated with the use of pathogenic, slow-growing M. tuberculosis. Collectively, these studies provide new information on the mechanism of inhibition of MbtAtb by salicyl-AMS and eight analogues, afford new SAR insights for these inhibitors, and highlight several suitable candidates for future preclinical evaluation.


Assuntos
Adenosina/análogos & derivados , Antituberculosos/farmacologia , Ligases/antagonistas & inibidores , Sideróforos/farmacologia , Adenosina/química , Adenosina/metabolismo , Adenosina/farmacologia , Antituberculosos/química , Antituberculosos/metabolismo , Bacillus subtilis/enzimologia , Desenho de Drogas , Escherichia coli/genética , Cinética , Ligases/química , Ligases/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Ligação Proteica , Sideróforos/química , Sideróforos/metabolismo , Relação Estrutura-Atividade
16.
J Plant Physiol ; 232: 216-225, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30537609

RESUMO

In plants, SIZ1 regulates abiotic and biotic stress responses by promoting the SUMOylation of proteins. The apple MdSIZ1 protein has conserved domains similar to those of Arabidopsis AtSIZ1. Real-time fluorescent quantitative analysis showed that MdSIZ1 gene expression was induced by phosphate-deficient conditions. In addition, the level of SUMOylation was also significantly increased under these conditions. The MYB transcription factor MdPHR1 might be a target for the SUMO protein, which is a phosphorus starvation-dependent protein. Subsequently, an MdSIZ1 expression vector was constructed and transformed in Arabidopsis mutant siz1-2 and apple callus. The MdSIZ1 transgenic Arabidopsis partially complemented the defect phenotype of siz1-2 under phosphate-deficient conditions. The survival rate, length of primary root, and number or density of lateral roots were similar between the transgenic lines and wild type (WT). Under phosphate-deficient conditions, the SUMO conjugate and fresh weight of the MdSIZ1 transgenic apple callus were improved compared with WT. The MdSIZ1 transgenic apple callus grew under phosphate-deficient conditions, whereas the MdSIZ1 sense apple callus did not. Therefore, MdSIZ1 is involved in the regulation of the phosphate-deficiency response in apple.


Assuntos
Ligases/fisiologia , Malus/fisiologia , Fosfatos/deficiência , Proteínas de Plantas/fisiologia , Arabidopsis , Regulação da Expressão Gênica de Plantas , Ligases/metabolismo , Malus/enzimologia , Malus/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sumoilação
17.
Biosci Biotechnol Biochem ; 83(4): 589-597, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30574825

RESUMO

Peptides, biologically occurring oligomers of amino acids linked by amide bonds, are essential for living organisms. Many peptides isolated as natural products have biological functions such as antimicrobial, antivirus and insecticidal activities. Peptides often possess structural features or modifications not found in proteins, including the presence of nonproteinogenic amino acids, macrocyclic ring formation, heterocyclization, N-methylation and decoration by sugars or acyl groups. Nature employs various strategies to increase the structural diversity of peptides. Enzymes that modify peptides to yield mature natural products are of great interest for discovering new enzyme chemistry and are important for medicinal chemistry applications. We have discovered novel peptide modifying enzymes and have identified: (i) a new class of amide bond forming-enzymes; (ii) a pathway to biosynthesize a carbonylmethylene-containing pseudodipeptide structure; and (iii) two distinct peptide epimerases. In this review, an overview of our findings on peptide modifying enzymes is presented.


Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/metabolismo , Ligases/metabolismo , Biossíntese Peptídica , Processamento de Proteína Pós-Traducional , Racemases e Epimerases/metabolismo , Acilação , Aminoácidos Cíclicos , Proteínas de Bactérias/isolamento & purificação , Biocatálise , Produtos Biológicos/química , Química Farmacêutica , Ciclização , Humanos , Ligases/isolamento & purificação , Metilação , Peptídeos/química , Peptídeos/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Racemases e Epimerases/isolamento & purificação
18.
Proc Natl Acad Sci U S A ; 115(39): 9797-9802, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30201715

RESUMO

Metabolically quiescent bacteria represent a large proportion of those in natural and host environments, and they are often refractory to antibiotic treatment. Such drug tolerance is also observed in the laboratory during stationary phase, when bacteria face stress and starvation-induced growth arrest. Tolerance requires (p)ppGpp signaling, which mediates the stress and starvation stringent response (SR), but the downstream effectors that confer tolerance are unclear. We previously demonstrated that the SR is linked to increased antioxidant defenses in Pseudomonas aeruginosa We now demonstrate that superoxide dismutase (SOD) activity is a key factor in SR-mediated multidrug tolerance in stationary-phase P. aeruginosa Inactivation of the SR leads to loss of SOD activity and decreased multidrug tolerance during stationary phase. Genetic or chemical complementation of SOD activity of the ΔrelA spoT mutant (ΔSR) is sufficient to restore antibiotic tolerance to WT levels. Remarkably, we observe high membrane permeability and increased drug internalization upon ablation of SOD activity. Combined, our results highlight an unprecedented mode of SR-mediated multidrug tolerance in stationary-phase P. aeruginosa and suggest that inhibition of SOD activity may potentiate current antibiotics.


Assuntos
Farmacorresistência Bacteriana Múltipla , Pseudomonas aeruginosa/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Antibacterianos/farmacologia , Relação Dose-Resposta a Droga , Gentamicinas/farmacologia , Ligases/metabolismo , Meropeném , Testes de Sensibilidade Microbiana , Ofloxacino/farmacologia , Pseudomonas aeruginosa/enzimologia , Transdução de Sinais , Superóxido Dismutase/fisiologia , Tienamicinas/farmacologia
19.
Proc Natl Acad Sci U S A ; 115(25): 6446-6451, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29866825

RESUMO

When faced with amino acid starvation, prokaryotic cells induce a stringent response that modulates their physiology. The stringent response is manifested by production of signaling molecules guanosine 5'-diphosphate,3'-diphosphate (ppGpp) and guanosine 5'-triphosphate,3'-diphosphate (pppGpp) that are also called alarmones. In many species, alarmone levels are regulated by a multidomain bifunctional alarmone synthetase/hydrolase called Rel. In this enzyme, there is an ACT domain at the carboxyl region that has an unknown function; however, similar ACT domains are present in other enzymes that have roles in controlling amino acid metabolism. In many cases, these other ACT domains have been shown to allosterically regulate enzyme activity through the binding of amino acids. Here, we show that the ACT domain present in the Rhodobacter capsulatus Rel alarmone synthetase/hydrolase binds branched-chain amino acids valine and isoleucine. We further show that the binding of these amino acids stimulates alarmone hydrolase activity both in vitro and in vivo. Furthermore, we found that the ACT domain present in Rel proteins from many diverse species also binds branched-chain amino acids. These results indicate that the cellular concentration of amino acids can directly affect Rel alarmone synthetase/hydrolase activity, thus adding another layer of control to current models of cellular control of the stringent response.


Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Hidrolases/metabolismo , Ligases/metabolismo , Rhodobacter capsulatus/metabolismo
20.
Molecules ; 23(6)2018 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-29882808

RESUMO

Polygonum minus (syn. Persicaria minor) is a herbal plant that is well known for producing sesquiterpenes, which contribute to its flavour and fragrance. This study describes the cloning and functional characterisation of PmSTPS1 and PmSTPS2, two sesquiterpene synthase genes that were identified from P. minus transcriptome data mining. The full-length sequences of the PmSTPS1 and PmSTPS2 genes were expressed in the E. coli pQE-2 expression vector. The sizes of PmSTPS1 and PmSTPS2 were 1098 bp and 1967 bp, respectively, with open reading frames (ORF) of 1047 and 1695 bp and encoding polypeptides of 348 and 564 amino acids, respectively. The proteins consist of three conserved motifs, namely, Asp-rich substrate binding (DDxxD), metal binding residues (NSE/DTE), and cytoplasmic ER retention (RxR), as well as the terpene synthase family N-terminal domain and C-terminal metal-binding domain. From the in vitro enzyme assays, using the farnesyl pyrophosphate (FPP) substrate, the PmSTPS1 enzyme produced multiple acyclic sesquiterpenes of β-farnesene, α-farnesene, and farnesol, while the PmSTPS2 enzyme produced an additional nerolidol as a final product. The results confirmed the roles of PmSTPS1 and PmSTPS2 in the biosynthesis pathway of P. minus, to produce aromatic sesquiterpenes.


Assuntos
Ligases/metabolismo , Polygonum/enzimologia , Sesquiterpenos/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Genes de Plantas , Ligases/química , Ligases/genética , Malásia , Fases de Leitura Aberta , Filogenia , Polygonum/genética , Homologia de Sequência de Aminoácidos
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