RESUMEN
Lipid biosynthesis in the pathogen Mycobacterium tuberculosis depends on biotin for posttranslational modification of key enzymes. However, the mycobacterial biotin synthetic pathway is not fully understood. Here, we show that rv1590, a gene of previously unknown function, is required by M. tuberculosis to synthesize biotin. Chemical-generic interaction experiments mapped the function of rv1590 to the conversion of dethiobiotin to biotin, which is catalyzed by biotin synthases (BioB). Biochemical studies confirmed that in contrast to BioB of Escherichia coli, BioB of M. tuberculosis requires Rv1590 (which we named "biotin synthase auxiliary protein" or BsaP), for activity. We found homologs of bsaP associated with bioB in many actinobacterial genomes, and confirmed that BioB of Mycobacterium smegmatis also requires BsaP. Structural comparisons of BsaP-associated biotin synthases with BsaP-independent biotin synthases suggest that the need for BsaP is determined by the [2Fe-2S] cluster that inserts sulfur into dethiobiotin. Our findings open new opportunities to seek BioB inhibitors to treat infections with M. tuberculosis and other pathogens.
Asunto(s)
Proteínas Bacterianas , Biotina , Mycobacterium tuberculosis , Biotina/metabolismo , Biotina/análogos & derivados , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Sulfurtransferasas/metabolismo , Sulfurtransferasas/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/enzimología , Escherichia coli/metabolismo , Escherichia coli/genéticaRESUMEN
DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from Mycobacterium tuberculosis (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.
Asunto(s)
Antituberculosos , Inhibidores Enzimáticos , Mycobacterium tuberculosis , Sintasas Poliquetidas , Bibliotecas de Moléculas Pequeñas , Tioléster Hidrolasas , Animales , Humanos , Ratones , Antituberculosos/química , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/efectos de los fármacos , Sintasas Poliquetidas/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Tioléster Hidrolasas/antagonistas & inhibidores , Tioléster Hidrolasas/metabolismo , Tioléster Hidrolasas/química , Tioléster Hidrolasas/genética , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
YisK is an uncharacterized protein in Bacillus subtilis previously shown to interact genetically with the elongasome protein Mbl. YisK overexpression leads to cell widening and lysis, phenotypes that are dependent on mbl and suppressed by mbl mutations. In the present work, we characterize YisK's localization, structure, and enzymatic activity. We show that YisK localizes as puncta that depend on Mbl. YisK belongs to the fumarylacetoacetate hydrolase (FAH) superfamily, and crystal structures revealed close structural similarity to two oxaloacetate (OAA) decarboxylases: human mitochondrial FAHD1 and Corynebacterium glutamicum Cg1458. We demonstrate that YisK can also catalyze the decarboxylation of OAA (K m = 134 µM, K cat = 31 min-1). A catalytic dead variant (YisK E148A, E150A) retains wild-type localization and still widens cells following overexpression, indicating these activities are not dependent on YisK catalysis. Conversely, a non-localizing variant (YisK E30A) retains wild-type enzymatic activity in vitro but localizes diffusely and no longer widens cells following overexpression. Together, these results suggest that YisK may be subject to spatial regulation that depends on the cell envelope synthesis machinery. IMPORTANCE The elongasome is a multiprotein complex that guides lengthwise growth in some bacteria. We previously showed that, in B. subtilis, overexpression of an uncharacterized putative enzyme (YisK) perturbed function of the actin-like elongasome protein Mbl. Here, we show that YisK exhibits Mbl-dependent localization. Through biochemical and structural characterization, we demonstrate that, like its mitochondrial homolog FAHD1, YisK can catalyze the decarboxylation of the oxaloacetate to pyruvate and CO2. YisK is the first example of an enzyme implicated in central carbon metabolism with subcellular localization that depends on Mbl.
Asunto(s)
Bacillus subtilis , Carboxiliasas , Humanos , Bacillus subtilis/metabolismo , Carboxiliasas/genética , Ácido Pirúvico , Oxaloacetatos , Hidrolasas/genéticaRESUMEN
Alternative mode-of-inhibition of clinically validated targets is an effective strategy for circumventing existing clinical drug resistance. Herein, we report 1,3-diarylpyrazolyl-acylsulfonamides as potent inhibitors of HadAB/BC, a 3-hydroxyl-ACP dehydratase complex required to iteratively elongate the meromycolate chain of mycolic acids in Mycobacterium tuberculosis (Mtb). Mutations in compound 1-resistant Mtb mutants mapped to HadC (Rv0637; K157R), while chemoproteomics confirmed the compound's binding to HadA (Rv0635), HadB (Rv0636), and HadC. The compounds effectively inhibited the HadAB and HadBC enzyme activities and affected mycolic acid biosynthesis in Mtb, in a concentration-dependent manner. Unlike known 3-hydroxyl-ACP dehydratase complex inhibitors of clinical significance, isoxyl and thioacetazone, 1,3-diarylpyrazolyl-acylsulfonamides did not require activation by EthA and thus are not liable to EthA-mediated resistance. Further, the crystal structure of a key compound in a complex with Mtb HadAB revealed unique binding interactions within the active site of HadAB, providing a useful tool for further structure-based optimization of the series.
Asunto(s)
Mycobacterium tuberculosis , Tioacetazona , Proteínas Bacterianas/metabolismo , Ácidos Micólicos/química , Tioacetazona/metabolismo , Tioacetazona/farmacología , Hidroliasas/química , Hidroliasas/metabolismo , Hidroliasas/farmacologíaRESUMEN
Diacylglycerol (DAG) is a versatile lipid whose 1,2-sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid. Effector proteins translocate to available DAG pools in the membranes by using conserved homology 1 (C1) domains as DAG-sensing modules. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC) as the first member of the DAG effector cohort. Herein, we report the high-resolution crystal structures of a C1 domain (C1B from PKCδ) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins.
Asunto(s)
Diglicéridos , Proteína Quinasa C , Animales , Membrana Celular/metabolismo , Diglicéridos/química , Unión Proteica , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , RatasRESUMEN
The ability of Mycobacterium tuberculosis (Mtb) to resist and tolerate antibiotics complicates the development of improved tuberculosis (TB) chemotherapies. Here we define the Mtb protein CinA as a major determinant of drug tolerance and as a potential target to shorten TB chemotherapy. By reducing the fraction of drug-tolerant persisters, genetic inactivation of cinA accelerated killing of Mtb by four antibiotics in clinical use: isoniazid, ethionamide, delamanid and pretomanid. Mtb ΔcinA was killed rapidly in conditions known to impede the efficacy of isoniazid, such as during nutrient starvation, during persistence in a caseum mimetic, in activated macrophages and during chronic mouse infection. Deletion of CinA also increased in vivo killing of Mtb by BPaL, a combination of pretomanid, bedaquiline and linezolid that is used to treat highly drug-resistant TB. Genetic and drug metabolism studies suggest that CinA mediates drug tolerance via cleavage of NAD-drug adducts.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Tolerancia a Medicamentos , Isoniazida/farmacología , Ratones , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
The isocitrate lyase paralogs of Mycobacterium tuberculosis (ICL1 and 2) are essential for mycobacterial persistence and constitute targets for the development of antituberculosis agents. We report that (2R,3S)-2-hydroxy-3-(nitromethyl)succinic acid (5-NIC) undergoes apparent retro-aldol cleavage as catalyzed by ICL1 to produce glyoxylate and 3-nitropropionic acid (3-NP), the latter of which is a covalent-inactivating agent of ICL1. Kinetic analysis of this reaction identified that 5-NIC serves as a robust and efficient mechanism-based inactivator of ICL1 (kinact/KI = (1.3 ± 0.1) × 103 M-1 s-1) with a partition ratio <1. Using enzyme kinetics, mass spectrometry, and X-ray crystallography, we identified that the reaction of the 5-NIC-derived 3-NP with the Cys191 thiolate of ICL1 results in formation of an ICL1-thiohydroxamate adduct as predicted. One aspect of the design of 5-NIC was to lower its overall charge compared to isocitrate to assist with cell permeability. Accordingly, the absence of the third carboxylate group will simplify the synthesis of pro-drug forms of 5-NIC for characterization in cell-infection models of M. tuberculosis.
Asunto(s)
Inhibidores Enzimáticos/química , Isocitratoliasa/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Succinatos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Glioxilatos/química , Glioxilatos/metabolismo , Isocitratoliasa/química , Isocitratoliasa/metabolismo , Cinética , Modelos Químicos , Nitrocompuestos/química , Nitrocompuestos/metabolismo , Propionatos/química , Propionatos/metabolismo , Unión Proteica , Succinatos/síntesis química , Succinatos/metabolismoRESUMEN
Optimal phage propagation depends on the regulation of the lysis of the infected host cell. In T4 phage infection, lysis occurs when the holin protein (T) forms lesions in the host membrane. However, the lethal function of T can be blocked by an antiholin (RI) during lysis inhibition (LIN). LIN sets if the infected cell undergoes superinfection, then the lysis is delayed until host/phage ratio becomes more favorable for the release of progeny. It has been thought that a signal derived from the superinfection is required to activate RI. Here we report structures that suggest a radically different model in which RI binds to T irrespective of superinfection, causing it to accumulate in a membrane as heterotetrameric 2RI-2T complex. Moreover, we show the complex binds non-specifically to DNA, suggesting that the gDNA from the superinfecting phage serves as the LIN signal and that stabilization of the complex by DNA binding is what defines LIN. Finally, we show that soluble domain of free RI crystallizes in a domain-swapped homotetramer, which likely works as a sink for RI molecules released from the RI-T complex to ensure efficient lysis. These results constitute the first structural basis and a new model not only for the historic LIN phenomenon but also for the temporal regulation of phage lysis in general.
Asunto(s)
Bacteriófago T4/fisiología , ADN Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Fenómenos Fisiológicos Bacterianos , Bacteriólisis , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Dominios ProteicosRESUMEN
Bacillus subtilis is a bacterium capable of differentiating into a spore form more resistant to environmental stress. Early in sporulation, each cell possesses two copies of a circular chromosome. A polar FtsZ ring (Z ring) directs septation over one of the chromosomes, generating two cell compartments. The smaller "forespore" compartment initially contains only 25 to 30% of one chromosome, and this transient genetic asymmetry is required for differentiation. Timely assembly of polar Z rings and precise capture of the chromosome in the forespore both require the DNA-binding protein RefZ. To mediate its role in chromosome capture, RefZ must bind to specific DNA motifs (RBMs) that localize near the poles at the time of septation. Cells artificially induced to express RefZ during vegetative growth cannot assemble Z rings, an effect that also requires DNA binding. We hypothesized that RefZ-RBM complexes mediate precise chromosome capture by modulating FtsZ function. To investigate, we isolated 10 RefZ loss-of-function (rLOF) variants unable to inhibit cell division yet still capable of binding RBMs. Sporulating cells expressing the rLOF variants in place of wild-type RefZ phenocopied a ΔrefZ mutant, suggesting that RefZ acts through an FtsZ-dependent mechanism. The crystal structure of RefZ was solved, and wild-type RefZ and the rLOF variants were further characterized. Our data suggest that RefZ's oligomerization state and specificity for the RBMs are critical determinants influencing RefZ's ability to affect FtsZ dynamics. We propose that RBM-bound RefZ complexes function as a developmentally regulated nucleoid occlusion system for fine-tuning the position of the septum relative to the chromosome during sporulation.IMPORTANCE The bacterial nucleoid forms a large, highly organized structure. Thus, in addition to storing the genetic code, the nucleoid harbors positional information that can be leveraged by DNA-binding proteins to spatially constrain cellular activities. During B. subtilis sporulation, the nucleoid undergoes reorganization, and the cell division protein FtsZ assembles polarly to direct septation over one chromosome. The TetR family protein RefZ binds DNA motifs (RBMs) localized near the poles at the time of division and is required for both timely FtsZ assembly and precise capture of DNA in the future spore compartment. Our data suggest that RefZ exploits nucleoid organization by associating with polarly localized RBMs to modulate the positioning of FtsZ relative to the chromosome during sporulation.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Esporas Bacterianas/crecimiento & desarrollo , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , División Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Esporas Bacterianas/química , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismoRESUMEN
Trichoderma spp. have been characterized for their capacity to act as biological control agents against several pathogens through the activity of secondary metabolites and cell wall degrading enzymes. However, only T. reesei has been widely studied for the ability to assimilate lignocellulose substrates. Protein analysis by SDS-PAGE of culture filtrate of T. virens revealed the presence of an unknown â¼77â¯kDa band protein (GLX1) that showed sequence homology to glyoxal-like oxidase genes involved in lignin degradation. The analysis and biochemical characterization of the 1,119 amino acid coded protein showed the presence of five carbohydrate-binding modules (CBMs) with affinity for colloidal chitin, and a functional glyoxal oxidase catalytic domain that is involved in the production of hydrogen peroxide when methylglyoxal was used as a substrate. The silencing of the glx1 gene resulted in mutants with more than 90% expression reduction and the absence of glyoxal oxidase catalytic activity. These mutants showed delayed hyphal growth, reduced colony and conidial hydrophobicity, but showed no changes in their biocontrol ability. Most significantly, mutants exhibited a loss of growth directionality resulting in a curled phenotype that was eliminated in the presence of exogenous H2O2. Here we present evidence that in T. virens, glx1 is not involved in the breakdown of lignin but instead is responsible for normal hyphal growth and morphology and likely does this through free radical production within the fungal cell wall. This is the first time that a glyoxal oxidase protein has been isolated and characterized in ascomycete fungi.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Pared Celular/enzimología , Proteínas Fúngicas/metabolismo , Hifa/crecimiento & desarrollo , Hifa/metabolismo , Trichoderma/enzimología , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/aislamiento & purificación , Secuencia de Aminoácidos/genética , Dominio Catalítico/fisiología , Cobre/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Silenciador del Gen , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Lignina/metabolismo , Fenotipo , Homología de Secuencia , Esporas Fúngicas/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1006939.].
RESUMEN
Upon inhibition of respiration, which occurs in hypoxic or nitric oxide-containing host microenvironments, Mycobacterium tuberculosis (Mtb) adopts a non-replicating "quiescent" state and becomes relatively unresponsive to antibiotic treatment. We used comprehensive mutant fitness analysis to identify regulatory and metabolic pathways that are essential for the survival of quiescent Mtb. This genetic study identified a protein acetyltransferase (Mt-Pat/Rv0998) that promoted survival and altered the flux of carbon from oxidative to reductive tricarboxylic acid (TCA) reactions. Reductive TCA requires malate dehydrogenase (MDH) and maintains the redox state of the NAD+/NADH pool. Genetic or chemical inhibition of MDH resulted in rapid cell death in both hypoxic cultures and in murine lung. These phenotypic data, in conjunction with significant structural differences between human and mycobacterial MDH enzymes that could be exploited for drug development, suggest a new strategy for eradicating quiescent bacteria.
Asunto(s)
Hipoxia/metabolismo , Lisina Acetiltransferasas/metabolismo , Mycobacterium tuberculosis/enzimología , Animales , Antibacterianos/química , Antibacterianos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Hipoxia/tratamiento farmacológico , Hipoxia/genética , Lisina Acetiltransferasas/antagonistas & inhibidores , Lisina Acetiltransferasas/genética , Ratones , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismoRESUMEN
Human infection by Mycobacterium tuberculosis (Mtb) continues to be a global epidemic. Computer-aided drug design (CADD) methods are used to accelerate traditional drug discovery efforts. One noncovalent interaction that is being increasingly identified in biological systems but is neglected in CADD is the anion-π interaction. The study reported herein supports the conclusion that anion-π interactions play a central role in directing the binding of phenyl-diketo acid (PDKA) inhibitors to malate synthase (GlcB), an enzyme required for Mycobacterium tuberculosis virulence. Using density functional theory methods (M06-2X/6-31+G(d)), a GlcB active site template was developed for a predictive model through a comparative analysis of PDKA-bound GlcB crystal structures. The active site model includes the PDKA molecule and the protein determinants of the electrostatic, hydrogen-bonding, and anion-π interactions involved in binding. The predictive model accurately determines the Asp 633-PDKA structural position upon binding and precisely predicts the relative binding enthalpies of a series of 2-ortho halide-PDKAs to GlcB. A screening model was also developed to efficiently assess the propensity of each PDKA analog to participate in an anion-π interaction; this method is in good agreement with both the predictive model and the experimental binding enthalpies for the 2-ortho halide-PDKAs. With the screening and predictive models in hand, we have developed an efficient method for computationally screening and evaluating the binding enthalpy of variously substituted PDKA molecules. This study serves to illustrate the contribution of this overlooked interaction to binding affinity and demonstrates the importance of integrating anion-π interactions into structure-based CADD.
Asunto(s)
Antituberculosos/farmacología , Malato Sintasa/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Antituberculosos/química , Sitios de Unión , Simulación por Computador , Malato Sintasa/metabolismo , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
Once considered a phenotypically monomorphic bacterium, there is a growing body of work demonstrating heterogeneity among Mycobacterium tuberculosis (Mtb) strains in clinically relevant characteristics, including virulence and response to antibiotics. However, the genetic and molecular basis for most phenotypic differences among Mtb strains remains unknown. To investigate the basis of strain variation in Mtb, we performed genome-wide transposon mutagenesis coupled with next-generation sequencing (TnSeq) for a panel of Mtb clinical isolates and the reference strain H37Rv to compare genetic requirements for in vitro growth across these strains. We developed an analytic approach to identify quantitative differences in genetic requirements between these genetically diverse strains, which vary in genomic structure and gene content. Using this methodology, we found differences between strains in their requirements for genes involved in fundamental cellular processes, including redox homeostasis and central carbon metabolism. Among the genes with differential requirements were katG, which encodes the activator of the first-line antitubercular agent isoniazid, and glcB, which encodes malate synthase, the target of a novel small-molecule inhibitor. Differences among strains in their requirement for katG and glcB predicted differences in their response to these antimicrobial agents. Importantly, these strain-specific differences in antibiotic response could not be predicted by genetic variants identified through whole genome sequencing or by gene expression analysis. Our results provide novel insight into the basis of variation among Mtb strains and demonstrate that TnSeq is a scalable method to predict clinically important phenotypic differences among Mtb strains.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Tuberculosis/genética , Elementos Transponibles de ADN , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mycobacterium tuberculosis/clasificación , Fenotipo , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Secuenciación Completa del GenomaRESUMEN
Fragment screening and high throughput screening are complementary approaches that combine with structural biology to explore the binding capabilities of an active site. We have used a fragment-based approach on malate synthase (GlcB) from Mycobacterium tuberculosis and discovered several novel binding chemotypes. In addition, the crystal structures of GlcB in complex with these fragments indicated conformational changes in the active site that represent the enzyme conformations during catalysis. Additional structures of the complex with malate and of the apo form of GlcB supported that hypothesis. Comparative analysis of GlcB structures in complex with 18 fragments allowed us to characterize the preferred chemotypes and their binding modes. The fragment structures showed a hydrogen bond to the backbone carbonyl of Met-631. We successfully incorporated an indole group from a fragment into an existing phenyl-diketo acid series. The resulting indole-containing inhibitor was 100-fold more potent than the parent phenyl-diketo acid with an IC50 value of 20 nm.
Asunto(s)
Malato Sintasa/química , Malato Sintasa/metabolismo , Malatos/metabolismo , Mycobacterium tuberculosis/enzimología , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.
Asunto(s)
Proteínas Bacterianas/química , Mycobacterium tuberculosis/química , Proteínas de Unión a las Penicilinas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carboxipeptidasas/química , Carboxipeptidasas/genética , Carboxipeptidasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Evolución Molecular Dirigida , Proteínas de Escherichia coli/química , Meropenem , Modelos Moleculares , Mutación , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Filogenia , Conformación Proteica , Tienamicinas/química , Tienamicinas/metabolismoRESUMEN
The glyoxylate shunt plays an important role in fatty acid metabolism and has been shown to be critical to survival of several pathogens involved in chronic infections. For Mycobacterium tuberculosis (Mtb), a strain with a defective glyoxylate shunt was previously shown to be unable to establish infection in a mouse model. We report the development of phenyl-diketo acid (PDKA) inhibitors of malate synthase (GlcB), one of two glyoxylate shunt enzymes, using structure-based methods. PDKA inhibitors were active against Mtb grown on acetate, and overexpression of GlcB ameliorated this inhibition. Crystal structures of complexes of GlcB with PDKA inhibitors guided optimization of potency. A selected PDKA compound demonstrated efficacy in a mouse model of tuberculosis. The discovery of these PDKA derivatives provides chemical validation of GlcB as an attractive target for tuberculosis therapeutics.