RESUMO
Pyruvate:quinone oxidoreductase (PQO) is a flavin-containing peripheral membrane enzyme catalyzing the decarboxylation of pyruvate to acetate and CO2 with quinone as an electron acceptor. Here, we investigate PQO activity in Corynebacterium glutamicum, examine purified PQO, and describe the crystal structure of the native enzyme and a truncated version. The specific PQO activity was highest in stationary phase cells grown in complex medium, lower in cells grown in complex medium containing glucose or acetate, and lowest in cells grown in minimal acetate-medium. A similar pattern with about 30-fold higher specific PQO activities was observed in C. glutamicum with plasmid-bound pqo expression under the control of the tac promoter, indicating that the differences in PQO activity are likely due to post-transcriptional control. Continuous cultivation of C. glutamicum at dilution rates between 0.05 and 0.4 h-1 revealed a negative correlation between PQO activity and growth rate. Kinetic analysis of PQO enzymes purified from cells grown in complex or in minimal acetate-medium revealed substantial differences in specific activity (72.3 vs. 11.9 U·mg protein-1) and turnover number (kcat: 440 vs. 78 s-1, respectively), suggesting post-translational modifications affecting PQO activity. Structural analysis of PQO revealed a homotetrameric arrangement very similar to the Escherichia coli pyruvate oxidase PoxB except for the C-terminal membrane binding domain, which exhibited a conformation markedly different from its PoxB counterpart. A truncated PQO variant lacking 17 C-terminal amino acids showed higher affinity to pyruvate and was independent of detergent activation, highlighting the importance of the C-terminus for enzyme activation and lipid binding.
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MbtI from Mycobacterium tuberculosis (Mtb) is a Mg2+-dependent salicylate synthase, belonging to the chorismate-utilizing enzyme (CUE) family. As a fundamental player in iron acquisition, MbtI promotes the survival and pathogenicity of Mtb in the infected host. Hence, it has emerged in the last decade as an innovative, potential target for the anti-virulence therapy of tuberculosis. In this context, 5-phenylfuran-2-carboxylic acids have been identified as potent MbtI inhibitors. The first co-crystal structure of MbtI in complex with a member of this class was described in 2020, showing the enzyme adopting an open configuration. Due to the high mobility of the loop adjacent to the binding pocket, large portions of the amino acid chain were not defined in the electron density map, hindering computational efforts aimed at structure-driven ligand optimization. Herein, we report a new, high-resolution co-crystal structure of MbtI with a furan-based derivative, in which the closed configuration of the enzyme allowed tracing the entirety of the active site pocket in the presence of the bound inhibitor. Moreover, we describe a new crystal structure of MbtI in open conformation and in complex with the known inhibitor methyl-AMT, suggesting that in vitro potency is not related to the observed enzyme conformation. These findings will prove fundamental to enhance the potency of this series via rational structure-based drug-design approaches.
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Actinobacteria possess unique ways to regulate the oxoglutarate metabolic node. Contrary to most organisms in which three enzymes compose the 2-oxoglutarate dehydrogenase complex (ODH), actinobacteria rely on a two-in-one protein (OdhA) in which both the oxidative decarboxylation and succinyl transferase steps are carried out by the same polypeptide. Here we describe high-resolution cryo-EM and crystallographic snapshots of representative enzymes from Mycobacterium smegmatis and Corynebacterium glutamicum, showing that OdhA is an 800-kDa homohexamer that assembles into a three-blade propeller shape. The obligate trimeric and dimeric states of the acyltransferase and dehydrogenase domains, respectively, are critical for maintaining the overall assembly, where both domains interact via subtle readjustments of their interfaces. Complexes obtained with substrate analogues, reaction products and allosteric regulators illustrate how these domains operate. Furthermore, we provide additional insights into the phosphorylation-dependent regulation of this enzymatic machinery by the signalling protein OdhI.
Assuntos
Corynebacterium glutamicum , Complexo Cetoglutarato Desidrogenase , Complexo Cetoglutarato Desidrogenase/metabolismo , Microscopia Crioeletrônica , Fosforilação , Corynebacterium glutamicum/metabolismoRESUMO
In Corynebacterium glutamicum the protein kinase PknG phosphorylates OdhI and thereby abolishes the inhibition of 2-oxoglutarate dehydrogenase activity by unphosphorylated OdhI. Our previous studies suggested that PknG activity is controlled by the periplasmic binding protein GlnH and the transmembrane protein GlnX, because ΔglnH and ΔglnX mutants showed a growth defect on glutamine similar to that of a ΔpknG mutant. We have now confirmed the involvement of GlnH and GlnX in the control of OdhI phosphorylation by analyzing the OdhI phosphorylation status and glutamate secretion in ΔglnH and ΔglnX mutants and by characterizing ΔglnX suppressor mutants. We provide evidence for GlnH being a lipoprotein and show by isothermal titration calorimetry that it binds l-aspartate and l-glutamate with moderate to low affinity, but not l-glutamine, l-asparagine, or 2-oxoglutarate. Based on a structural comparison with GlnH of Mycobacterium tuberculosis, two residues critical for the binding affinity were identified and verified. The predicted GlnX topology with four transmembrane segments and two periplasmic domains was confirmed by PhoA and LacZ fusions. A structural model of GlnX suggested that, with the exception of a poorly ordered N-terminal region, the entire protein is composed of α-helices and small loops or linkers, and it revealed similarities to other bacterial transmembrane receptors. Our results suggest that the GlnH-GlnX-PknG-OdhI-OdhA signal transduction cascade serves to adapt the flux of 2-oxoglutarate between ammonium assimilation via glutamate dehydrogenase and energy generation via the tricarboxylic acid (TCA) cycle to the availability of the amino group donors l-glutamate and l-aspartate in the environment. IMPORTANCE Actinobacteria comprise a large number of species playing important roles in biotechnology and medicine, such as Corynebacterium glutamicum, the major industrial amino acid producer, and Mycobacterium tuberculosis, the pathogen causing tuberculosis. Many actinobacteria use a signal transduction process in which the phosphorylation status of OdhI (corynebacteria) or GarA (mycobacteria) regulates the carbon flux at the 2-oxoglutarate node. Inhibition of 2-oxoglutarate dehydrogenase by unphosphorylated OdhI shifts the flux of 2-oxoglutarate from the TCA cycle toward glutamate formation and, thus, ammonium assimilation. Phosphorylation of OdhI/GarA is catalyzed by the protein kinase PknG, whose activity was proposed to be controlled by the periplasmic binding protein GlnH and the transmembrane protein GlnX. In this study, we combined genetic, biochemical, and structural modeling approaches to characterize GlnH and GlnX of C. glutamicum and confirm their roles in the GlnH-GlnX-PknG-OdhI-OdhA signal transduction cascade. These findings are relevant also to other Actinobacteria employing a similar control process.
Assuntos
Corynebacterium glutamicum , Mycobacterium tuberculosis , Proteínas Periplásmicas de Ligação , Fosforilação , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ácido Aspártico/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas Quinases/metabolismo , Mycobacterium tuberculosis/genética , Transdução de Sinais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Complexo Cetoglutarato Desidrogenase/genética , Complexo Cetoglutarato Desidrogenase/metabolismoRESUMO
Glutamate dehydrogenase (GDH) plays a key role in the metabolism of glutamate, an important compound at a cross-road of carbon and nitrogen metabolism and a relevant neurotransmitter. Despite being one of the first discovered allosteric enzymes, GDH still poses challenges for structural characterization of its allosteric sites. Only the structures with ADP, and at low (3.5 Å) resolution, are available for mammalian GDH complexes with allosteric activators. Here, we aim at deciphering a structural basis for the GDH allosteric activation using bovine GDH as a model. For the first time, we report a mammalian GDH structure in a ternary complex with the activators leucine and ADP, co-crystallized with potassium ion, resolved to 2.45 Å. An improved 2.4-angstrom resolution of the GDH complex with ADP is also presented. The ternary complex with leucine and ADP differs from the binary complex with ADP by the conformation of GDH C-terminus, involved in the leucine binding and subunit interactions. The potassium site, identified in this work, may mediate interactions between the leucine and ADP binding sites. Our data provide novel insights into the mechanisms of GDH activation by leucine and ADP, linked to the enzyme regulation by (de)acetylation.
Assuntos
Glutamato Desidrogenase , Ácido Glutâmico , Difosfato de Adenosina/metabolismo , Regulação Alostérica , Animais , Carbono , Bovinos , Glutamato Desidrogenase/metabolismo , Ácido Glutâmico/metabolismo , Leucina/metabolismo , Mamíferos/metabolismo , Nitrogênio , PotássioRESUMO
Type VIIb secretion systems (T7SSb) were recently proposed to mediate different aspects of Firmicutes physiology, including bacterial pathogenicity and competition. However, their architecture and mechanism of action remain largely obscure. Here, we present a detailed analysis of the T7SSb-mediated bacterial competition in Bacillus subtilis, using the effector YxiD as a model for the LXG secreted toxins. By systematically investigating protein-protein interactions, we reveal that the membrane subunit YukC contacts all T7SSb components, including the WXG100 substrate YukE and the LXG effector YxiD. YukC's crystal structure shows unique features, suggesting an intrinsic flexibility that is required for T7SSb antibacterial activity. Overall, our results shed light on the role and molecular organization of the T7SSb and demonstrate the potential of B. subtilis as a model system for extensive structure-function studies of these secretion machineries. IMPORTANCE Type VII secretion systems mediate protein extrusion from Gram-positive bacteria and are classified as T7SSa and T7SSb in Actinobacteria and in Firmicutes, respectively. Despite the genetic divergence of T7SSa and T7SSb, the high degree of structural similarity of their WXG100 substrates suggests similar secretion mechanisms. Recent advances revealed the structures of several T7SSa cytoplasmic membrane complexes, but the molecular mechanism of secretion and the T7SSb architecture remain obscure. Here, we provide hints on the organization of T7SSb in B. subtilis and a high-resolution structure of its central pseudokinase subunit, opening new perspectives for the understanding of the T7SSb secretion mechanism by using B. subtilis as an amenable bacterial model.
Assuntos
Sistemas de Secreção Tipo VII , Sistemas de Secreção Tipo VII/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/metabolismoRESUMO
α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high-molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an odhA gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.
Assuntos
Actinobacteria/metabolismo , Complexo Cetoglutarato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismo , Bactérias/metabolismo , Fenômenos Bioquímicos , Biologia Computacional , Cristalografia por Raios X , Cinética , Conformação Molecular , Mycobacterium tuberculosis/metabolismo , Plasmídeos/metabolismo , Ácido PirúvicoRESUMO
Signal transduction is essential for bacteria to adapt to changing environmental conditions. Among many forms of posttranslational modifications, reversible protein phosphorylation has evolved as a ubiquitous molecular mechanism of protein regulation in response to specific stimuli. The Ser/Thr protein kinase PknG modulates the fate of intracellular glutamate by controlling the phosphorylation status of the 2-oxoglutarate dehydrogenase regulator OdhI, a function that is conserved among diverse actinobacteria. PknG has a modular organization characterized by the presence of regulatory domains surrounding the catalytic domain. Here, we present an investigation using in vivo experiments, as well as biochemical and structural methods, of the molecular basis of the regulation of PknG from Corynebacterium glutamicum (CgPknG), in the light of previous knowledge available for the kinase from Mycobacterium tuberculosis (MtbPknG). We found that OdhI phosphorylation by CgPknG is regulated by a conserved mechanism that depends on a C-terminal domain composed of tetratricopeptide repeats (TPRs) essential for metabolic homeostasis. Furthermore, we identified a conserved structural motif that physically connects the TPR domain to a ß-hairpin within the flexible N-terminal region that is involved in docking interactions with OdhI. Based on our results and previous reports, we propose a model in which the TPR domain of PknG couples signal detection to the specific phosphorylation of OdhI. Overall, the available data indicate that conserved PknG domains in distant actinobacteria retain their roles in kinase regulation in response to nutrient availability. IMPORTANCE Bacteria control the metabolic processes by which they obtain nutrients and energy in order to adapt to the environment. Actinobacteria, one of the largest bacterial phyla of major importance for biotechnology, medicine, and agriculture, developed a unique control process that revolves around a key protein, the protein kinase PknG. Here, we use genetic, biochemical, and structural approaches to study PknG in a system that regulates glutamate production in Corynebacterium glutamicum, a species used for the industrial production of amino acids. The reported findings are conserved in related Actinobacteria, with broader significance for microorganisms that cause disease, as well as environmental species used industrially to produce amino acids and antibiotics every year.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Repetições de Tetratricopeptídeos , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Regulação Bacteriana da Expressão Gênica , Ácido Glutâmico/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Fosforilação , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Transdução de SinaisRESUMO
Mycobacterium tuberculosis, the etiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosis's pathogenic hallmarks is its ability to persist in a dormant state in the host. Thus, this pathogen has developed mechanisms to withstand stressful conditions found in the human host. Particularly, the Ser/Thr-protein kinase PknG has gained relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomic analyses of protein extracts from M. tuberculosis H37Rv and a mutant lacking pknG. We found that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance compared with the wild-type. The main biological processes affected by pknG deletion were cell envelope components biosynthesis and response to hypoxia. Thirteen DosR-regulated proteins were underrepresented in the pknG deletion mutant, including Hrp-1, which was 12.5-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, our results allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival. SIGNIFICANCE: PknG is a Ser/Thr kinase from Mycobacterium tuberculosis with key roles in bacterial metabolism and bacterial survival within the host. However, at present the molecular mechanisms underlying these functions remain largely unknown. In this work, we evaluate the effect of pknG deletion on M. tuberculosis proteome using different approaches. Our results clearly show that the global proteome was remodeled in the absence of PknG and shed light on new molecular mechanism underlying PknG role. Altogether, this work contributes to a better understanding of the molecular bases of the adaptation of M. tuberculosis, one of the most deadly human pathogens, to its host.
Assuntos
Fenômenos Biológicos , Mycobacterium tuberculosis , Proteínas de Bactérias/genética , Humanos , Hipóxia , Mycobacterium tuberculosis/genética , Proteínas Serina-Treonina Quinases/genética , Proteoma , ProteômicaRESUMO
The Mg2+-dependent Mycobacterium tuberculosis salicylate synthase (MbtI) is a key enzyme involved in the biosynthesis of siderophores. Because iron is essential for the survival and pathogenicity of the microorganism, this protein constitutes an attractive target for antitubercular therapy, also considering the absence of homologous enzymes in mammals. An extension of the structure-activity relationships of our furan-based candidates allowed us to disclose the most potent competitive inhibitor known to date (10, Ki = 4 µM), which also proved effective on mycobacterial cultures. By structural studies, we characterized its unexpected Mg2+-independent binding mode. We also investigated the role of the Mg2+ cofactor in catalysis, analyzing the first crystal structure of the MbtI-Mg2+-salicylate ternary complex. Overall, these results pave the way for the development of novel antituberculars through the rational design of improved MbtI inhibitors.
Assuntos
Desenho de Fármacos , Liases/química , Liases/metabolismo , Magnésio/metabolismo , Mycobacterium tuberculosis/enzimologia , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Phosphonate analogs of pyruvate and 2-oxoglutarate are established specific inhibitors of cognate 2-oxo acid dehydrogenases. The present work develops application of this class of compounds to specific in vivo inhibition of 2-oxoglutarate dehydrogenase (OGDH) and its isoenzyme, 2-oxoadipate dehydrogenase (OADH). The isoenzymes-enriched preparations from the rat tissues with different expression of OADH and OGDH are used to characterize their interaction with 2-oxoglutarate (OG), 2-oxoadipate (OA) and the phosphonate analogs. Despite a 100-fold difference in the isoenzymes ratio in the heart and liver, similar Michaelis saturations by OG are inherent in the enzyme preparations from these tissues ( K m O G = 0.45 ± 0.06 and 0.27 ± 0.026 mM, respectively), indicating no significant contribution of OADH to the OGDH reaction, or similar affinities of the isoenzymes to OG. However, the preparations differ in the catalysis of OADH reaction. The heart preparation, where OADH/OGDH ratio is ≈ 0.01, possesses low-affinity sites to OA ( K m O A = 0.55 ± 0.07 mM). The liver preparation, where OADH/OGDH ratio is ≈ 1.6, demonstrates a biphasic saturation with OA: the low-affinity sites ( K m , 2 O A = 0.45 ± 0.12 mM) are similar to those of the heart preparation; the high-affinity sites ( K m , 1 O A = 0.008 ± 0.001 mM), revealed in the liver preparation only, are attributed to OADH. Phosphonate analogs of C5-C7 dicarboxylic 2-oxo acids inhibit OGDH and OADH competitively to 2-oxo substrates in all sites. The high-affinity sites for OA are affected the least by the C5 analog (succinyl phosphonate) and the most by the C7 one (adipoyl phosphonate). The opposite reactivity is inherent in both the low-affinity OA-binding sites and OG-binding sites. The C6 analog (glutaryl phosphonate) does not exhibit a significant preference to either OADH or OGDH. Structural analysis of the phosphonates binding to OADH and OGDH reveals the substitution of a tyrosine residue in OGDH for a serine residue in OADH among structural determinants of the preferential binding of the bulkier ligands to OADH. The consistent kinetic and structural results expose adipoyl phosphonate as a valuable pharmacological tool for specific in vivo inhibition of the DHTKD1-encoded OADH, a new member of mammalian family of 2-oxo acid dehydrogenases, up-regulated in some cancers and associated with diabetes and obesity.
RESUMO
Mycobacterial KGD, the thiamine diphosphate (ThDP)-dependent E1o component of the 2-oxoglutarate dehydrogenase complex (OGDHC), is known to undergo significant conformational changes during catalysis with two distinct conformational states, previously named as the early and late state. In this work, we employ two phosphonate analogues of 2-oxoglutarate (OG), i.e. succinyl phosphonate (SP) and phosphono ethyl succinyl phosphonate (PESP), as tools to isolate the first catalytic steps and understand the significance of conformational transitions for the enzyme regulation. The kinetics showed a more efficient inhibition of mycobacterial E1o by SP (Ki 0.043⯱â¯0.013â¯mM) than PESP (Ki 0.88⯱â¯0.28â¯mM), consistent with the different circular dichroism spectra of the corresponding complexes. PESP allowed us to get crystallographic snapshots of the Michaelis-like complex, the first one for 2-oxo acid dehydrogenases, followed by the covalent adduction of the inhibitor to ThDP, mimicking the pre-decarboxylation complex. In addition, covalent ThDP-phosphonate complexes obtained with both compounds by co-crystallization were in the late conformational state, probably corresponding to slowly dissociating enzyme-inhibitor complexes. We discuss the relevance of these findings in terms of regulatory features of the mycobacterial E1o enzymes, and in the perspective of developing tools for species-specific metabolic regulation.
Assuntos
Complexo Cetoglutarato Desidrogenase/metabolismo , Mycobacterium/enzimologia , Domínio Catalítico , Complexo Cetoglutarato Desidrogenase/química , Ácidos Cetoglutáricos/metabolismo , Cinética , Mycobacterium/metabolismo , Organofosfonatos/metabolismo , Oxirredutases/metabolismo , Ligação Proteica , Succinatos/metabolismo , Tiamina Pirofosfato/metabolismoRESUMO
Protein phosphorylation is known to be one of the keystones of signal sensing and transduction in all living organisms. Once thought to be essentially confined to the eukaryotic kingdoms, reversible phosphorylation on serine, threonine and tyrosine residues, has now been shown to play a major role in many prokaryotes, where the number of Ser/Thr protein kinases (STPKs) equals or even exceeds that of two component systems. Mycobacterium tuberculosis, the etiological agent of tuberculosis, is one of the most studied organisms for the role of STPK-mediated signaling in bacteria. Driven by the interest and tractability of these enzymes as potential therapeutic targets, extensive studies revealed the remarkable conservation of protein kinases and their cognate phosphatases across evolution, and their involvement in bacterial physiology and virulence. Here, we present an overview of the current knowledge of mycobacterial STPKs structures and kinase activation mechanisms, and we then focus on PknB and PknG, two well-characterized STPKs that are essential for the intracellular survival of the bacillus. We summarize the mechanistic evidence that links PknB to the regulation of peptidoglycan synthesis in cell division and morphogenesis, and the major findings that establishes PknG as a master regulator of central carbon and nitrogen metabolism. Two decades after the discovery of STPKs in M. tuberculosis, the emerging landscape of O-phosphosignaling is starting to unveil how eukaryotic-like kinases can be engaged in unique, non-eukaryotic-like, signaling mechanisms in mycobacteria.
RESUMO
Forkhead-associated (FHA) domains are modules that bind to phosphothreonine (pThr) residues in signaling cascades. The FHA-containing mycobacterial protein GarA is a central element of a phosphorylation-dependent signaling pathway that redirects metabolic flux in response to amino acid starvation or cell growth requirements. GarA acts as a phosphorylation-dependent ON/OFF molecular switch. In its nonphosphorylated ON state, the GarA FHA domain engages in phosphorylation-independent interactions with various metabolic enzymes that orchestrate nitrogen flow, such as 2-oxoglutarate decarboxylase (KGD). However, phosphorylation at the GarA N-terminal region by the protein kinase PknB or PknG triggers autoinhibition through the intramolecular association of the N-terminal domain with the FHA domain, thus blocking all downstream interactions. To investigate these different FHA binding modes, we solved the crystal structures of the mycobacterial upstream (phosphorylation-dependent) complex PknB-GarA and the downstream (phosphorylation-independent) complex GarA-KGD. Our results show that the phosphorylated activation loop of PknB serves as a docking site to recruit GarA through canonical FHA-pThr interactions. However, the same GarA FHA-binding pocket targets an allosteric site on nonphosphorylated KGD, where a key element of recognition is a phosphomimetic aspartate. Further enzymatic and mutagenesis studies revealed that GarA acted as a dynamic allosteric inhibitor of KGD by preventing crucial motions in KGD that are necessary for catalysis. Our results provide evidence for physiological phosphomimetics, supporting numerous mutagenesis studies using such approaches, and illustrate how evolution can shape a single FHA-binding pocket to specifically interact with multiple phosphorylated and nonphosphorylated protein partners.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Mycobacterium tuberculosis/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Carboxiliases/química , Carboxiliases/genética , Carboxiliases/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Mutação , Mycobacterium tuberculosis/genética , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de ProteínaRESUMO
Protein phosphorylation is known to be one of the keystones of signal sensing and transduction in all living organisms. Once thought to be essentially confined to the eukaryotic kingdoms, reversible phosphorylation on serine, threonine, and tyrosine residues, has now been shown to play a major role in many prokaryotes, where the number of Ser/Thr protein kinases (STPKs) equals or even exceeds that of two-component systems. Mycobacterium tuberculosis, the etiological agent of tuberculosis, is one of the most studied organisms for the role of STPK-mediated signaling in bacteria. Driven by the interest and tractability of these enzymes as potential therapeutic targets, extensive studies revealed the remarkable conservation of protein kinases and their cognate phosphatases across evolution, and their involvement in bacterial physiology and virulence. Here, we present an overview of the current knowledge of mycobacterial STPK structures and kinase activation mechanisms, and we then focus on PknB and PknG, two well-characterized STPKs that are essential for the intracellular survival of the bacillus. We summarize the mechanistic evidence that links PknB to the regulation of peptidoglycan synthesis in cell division and morphogenesis, and the major findings that establish PknG as a master regulator of central carbon and nitrogen metabolism. Two decades after the discovery of STPKs in M. tuberculosis, the emerging landscape of O-phosphosignaling is starting to unveil how eukaryotic-like kinases can be engaged in unique, non-eukaryotic-like, signaling mechanisms in mycobacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ácido Glutâmico/metabolismo , Peptidoglicano/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Starting from the analysis of the hypothetical binding mode of our previous furan-based hit (I), we successfully achieved our objective to replace the nitro moiety, leading to the disclosure of a new lead exhibiting a strong activity against MbtI. Our best candidate 1 h displayed a Ki of 8.8 µM and its antimycobacterial activity (MIC99 = 250 µM) is conceivably related to mycobactin biosynthesis inhibition. These results support the hypothesis that 5-phenylfuran-2-carboxylic derivatives are a promising class of MbtI inhibitors.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Furanos/química , Liases/antagonistas & inibidores , Sítios de Ligação , Inibidores Enzimáticos/química , Liases/química , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mycobacterium bovis/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
PknG from Mycobacterium tuberculosis is a multidomain Serine/Threonine protein kinase that regulates bacterial metabolism as well as the pathogen's ability to survive inside the host by still uncertain mechanisms. To uncover PknG interactome we developed an affinity purification-mass spectrometry strategy to stepwise recover PknG substrates and interactors; and to identify those involving PknG autophosphorylated docking sites. We report a confident list of 7 new putative substrates and 66 direct or indirect partners indicating that PknG regulates many physiological processes, such as nitrogen and energy metabolism, cell wall synthesis and protein translation. GarA and the 50S ribosomal protein L13, two previously reported substrates of PknG, were recovered in our interactome. Comparative proteome analyses of wild type and pknG null mutant M. tuberculosis strains provided evidence that two kinase interactors, the FHA-domain containing protein GarA and the enzyme glutamine synthetase, are indeed endogenous substrates of PknG, stressing the role of this kinase in the regulation of nitrogen metabolism. Interestingly, a second FHA protein was identified as a PknG substrate. Our results show that PknG phosphorylates specific residues in both glutamine synthetase and FhaA in vitro, and suggest that these proteins are phosphorylated by PknG in living mycobacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Mutação , Mycobacterium tuberculosis/genética , Fosforilação , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Especificidade por SubstratoRESUMO
Sensing and response to changes in nutrient availability are essential for the lifestyle of environmental and pathogenic bacteria. Serine/threonine protein kinase G (PknG) is required for virulence of the human pathogen Mycobacterium tuberculosis, and its putative substrate GarA regulates the tricarboxylic acid cycle in M. tuberculosis and other Actinobacteria by protein-protein binding. We sought to understand the stimuli that lead to phosphorylation of GarA, and the roles of this regulatory system in pathogenic and non-pathogenic bacteria. We discovered that M. tuberculosis lacking garA was severely attenuated in mice and macrophages and furthermore that GarA lacking phosphorylation sites failed to restore the growth of garA deficient M. tuberculosis in macrophages. Additionally we examined the impact of genetic disruption of pknG or garA upon protein phosphorylation, nutrient utilization and the intracellular metabolome. We found that phosphorylation of GarA requires PknG and depends on nutrient availability, with glutamate and aspartate being the main stimuli. Disruption of pknG or garA caused opposing effects on metabolism: a defect in glutamate catabolism or depletion of intracellular glutamate, respectively. Strikingly, disruption of the phosphorylation sites of GarA was sufficient to recapitulate defects caused by pknG deletion. The results suggest that GarA is a cellular target of PknG and the metabolomics data demonstrate that the function of this signaling system is in metabolic regulation. This function in amino acid homeostasis is conserved amongst the Actinobacteria and provides an example of the close relationship between metabolism and virulence.
Assuntos
Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Metabolômica , Mycobacterium tuberculosis , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Ácido Aspártico/metabolismo , Proteínas de Bactérias/genética , Ácido Glutâmico/metabolismo , Homeostase , Macrófagos/microbiologia , Camundongos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Tuberculose/microbiologia , VirulênciaRESUMO
Eukaryotic-like Ser/Thr protein kinases (ePKs) have been identified in many bacterial species, where they are known to mediate signalling mechanisms that share several features with their eukaryotic counterparts. In Mycobacterium tuberculosis, PknI is one of the 11 predicted ePKs and it has been related to bacterial virulence. In order to better understand the molecular basis of its role in mycobacterial signalling, we solved the crystal structure of the PknI cytoplasmic domain. We found that even though PknI possesses most conserved elements characteristic of Hanks-type kinases, it is degraded in several motifs that are essential for the ePKs catalytic activity. Most notably, PknI presents a remarkably short activation segment lacking a peptide-substrate binding site. Consistent with this observation and similar to earlier findings for eukaryotic pseudokinases, no kinase activity was detected for the catalytic domain of PknI, against different substrates and in various experimental conditions. Based on these results, we conclude that PknI may rely on unconventional mechanism(s) for kinase activity and/or it could play alternative role(s) in mycobacterial signalling. DATABASE: Atomic coordinates and structure factors for the catalytic domain of M. tuberculosis PknI are in the Protein Data Bank under the accession codes 5M06 (wild-type PknI + ADP), 5M07 (PknI_C20A), 5M08 (PknI_C20A_R136A) and 5M09 (PknI_C20A_R136N).
Assuntos
Proteínas de Bactérias/química , Mycobacterium tuberculosis/química , Proteínas Serina-Treonina Quinases/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meromycolic chain of mycolic acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the mycolic acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the mycolic acid biosynthesis modulation and possible inhibition strategies for this promising drug target.