RESUMEN
The bacterial cell wall is a multi-layered mesh, whose major component is peptidoglycan (PG), a sugar polymer cross-linked by short peptide stems. During cell division, a careful balance of PG synthesis and degradation, precisely coordinated both in time and space, is necessary to prevent uncontrolled destruction of the cell wall. In Corynebacteriales, the D,L endopeptidase RipA has emerged as a major PG hydrolase for cell separation, and RipA defaults have major implications for virulence of the human pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. However, the precise mechanisms by which RipA mediates cell separation remain elusive. Here we report phylogenetic, biochemical, and structural analysis of the Corynebacterium glutamicum homologue of RipA, Cg1735. The crystal structures of full-length Cg1735 in two different crystal forms revealed the C-terminal NlpC/P60 catalytic domain obtruded by its N-terminal conserved coiled-coil domain, which locks the enzyme in an autoinhibited state. We show that this autoinhibition is relieved by the extracellular core domain of the transmembrane septal protein Cg1604. The crystal structure of Cg1604 revealed a (ß/α) protein with an overall topology similar to that of receiver domains from response regulator proteins. The atomic model of the Cg1735-Cg1604 complex, based on bioinformatical and mutational analysis, indicates that a conserved, distal-membrane helical insertion in Cg1604 is responsible for Cg1735 activation. The reported data provide important insights into how intracellular cell division signal(s), yet to be identified, control PG hydrolysis during RipA-mediated cell separation in Corynebacteriales.
Asunto(s)
Actinomycetales , Proteínas Bacterianas , Actinomycetales/citología , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/genética , Peptidoglicano/metabolismo , FilogeniaRESUMEN
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.
Asunto(s)
Actinobacteria/metabolismo , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismo , Bacterias/metabolismo , Fenómenos Bioquímicos , Biología Computacional , Cristalografía por Rayos X , Cinética , Conformación Molecular , Mycobacterium tuberculosis/metabolismo , Plásmidos/metabolismo , Ácido PirúvicoRESUMEN
Upon triggering by their inducer, signal transduction ATPases with numerous domains (STANDs), initially in monomeric resting forms, multimerize into large hubs that activate target macromolecules. This process requires conversion of the STAND conserved core (the NOD) from a closed form encasing an ADP molecule to an ATP-bound open form prone to multimerize. In the absence of inducer, autoinhibitory interactions maintain the NOD closed. In particular, in resting STAND proteins with an LRR- or WD40-type sensor domain, the latter establishes interactions with the NOD that are disrupted in the multimerization-competent forms. Here, we solved the first crystal structure of a STAND with a tetratricopeptide repeat sensor domain, PH0952 from Pyrococcus horikoshii, revealing analogous NOD-sensor contacts. We use this structural information to experimentally demonstrate that similar interactions also exist in a PH0952 homolog, the MalT STAND archetype, and actually contribute to the MalT autoinhibition in vitro and in vivo. We propose that STAND activation occurs by stepwise release of autoinhibitory contacts coupled to the unmasking of inducer-binding determinants. The MalT example suggests that STAND weak autoinhibitory interactions could assist the binding of inhibitory proteins by placing in register inhibitor recognition elements born by two domains.
Asunto(s)
Adenosina Trifosfatasas/química , Conformación Proteica , Dominios Proteicos/genética , Repeticiones de Tetratricopéptidos/genética , Adenosina Trifosfatasas/genética , Cristalografía por Rayos X , Escherichia coli/genética , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína/genética , Transducción de Señal/genética , Repeticiones WD40/genéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Ácido Glutámico/metabolismo , Peptidoglicano/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
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.
Asunto(s)
Complejo Cetoglutarato Deshidrogenasa/metabolismo , Mycobacterium/enzimología , Dominio Catalítico , Complejo Cetoglutarato Deshidrogenasa/química , Ácidos Cetoglutáricos/metabolismo , Cinética , Mycobacterium/metabolismo , Organofosfonatos/metabolismo , Oxidorreductasas/metabolismo , Unión Proteica , Succinatos/metabolismo , Tiamina Pirofosfato/metabolismoRESUMEN
Carbapenems are "last resort" ß-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MßLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different ß-lactams in all MßLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MßLs.
Asunto(s)
Antibacterianos/química , Ceftazidima/química , Imipenem/química , Meropenem/química , Zinc/química , beta-Lactamasas/química , Secuencia de Aminoácidos , Antibacterianos/metabolismo , Dominio Catalítico , Cefepima/química , Cefepima/metabolismo , Cefotaxima/química , Cefotaxima/metabolismo , Ceftazidima/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Imipenem/metabolismo , Cinética , Meropenem/metabolismo , Modelos Moleculares , Piperacilina/química , Piperacilina/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Zinc/metabolismo , Resistencia betalactámica , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
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.
Asunto(s)
Aminoácidos/metabolismo , Proteínas Bacterianas/metabolismo , Metabolómica , Mycobacterium tuberculosis , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Ácido Aspártico/metabolismo , Proteínas Bacterianas/genética , Ácido Glutámico/metabolismo , Homeostasis , Macrófagos/microbiología , Ratones , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Tuberculosis/microbiología , VirulenciaRESUMEN
The availability of whole-genome sequence data, made possible by significant advances in DNA sequencing technology, led to the emergence of structural genomics projects in the late 1990s. These projects not only significantly increased the number of 3D structures deposited in the Protein Data Bank in the last two decades, but also influenced present crystallographic strategies by introducing automation and high-throughput approaches in the structure-determination pipeline. Today, dedicated crystallization facilities, many of which are open to the general user community, routinely set up and track thousands of crystallization screening trials per day. Here, we review the current methods for high-throughput crystallization and procedures to obtain crystals suitable for X-ray diffraction studies, and we describe the crystallization pipeline implemented in the medium-scale crystallography platform at the Institut Pasteur (Paris) as an example.
Asunto(s)
Academias e Institutos , Biología Computacional , Bases de Datos de Proteínas , Cristalografía por Rayos X , HumanosRESUMEN
The transcriptional regulator CpxR mediates an adaptive response to envelope stress, tightly linked to virulence and antibiotics resistance in several Gammaproteobacteria pathogens. In this work, we integrated crystallographic and small-angle X-ray scattering data to gain insights into the structure and conformational plasticity of CpxR from Escherichia coli. CpxR dimerizes through two alternative interaction surfaces. Moreover, widely different CpxR conformations coexist in solution, from compact to fully extended ones. The possible functional implications of these structural features are discussed.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Gammaproteobacteria/metabolismo , Regulación Bacteriana de la Expresión Génica , Virulencia , Difracción de Rayos XRESUMEN
Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannoside, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of membrane-associated glycosyltransferases for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here, we determined that PimA preferentially binds to negatively charged phosphatidyl-myo-inositol substrate and non-substrate membrane model systems (small unilamellar vesicle) through its N-terminal domain, inducing an important structural reorganization of anionic phospholipids. By using a combination of single-point mutagenesis, circular dichroism, and a variety of fluorescence spectroscopy techniques, we determined that this interaction is mainly mediated by an amphipathic α-helix (α2), which undergoes a substantial conformational change and localizes in the vicinity of the negatively charged lipid headgroups and the very first carbon atoms of the acyl chains, at the PimA-phospholipid interface. Interestingly, a flexible region within the N-terminal domain, which undergoes ß-strand-to-α-helix and α-helix-to-ß-strand transitions during catalysis, interacts with anionic phospholipids; however, the effect is markedly less pronounced to that observed for the amphipathic α2, likely reflecting structural plasticity/variability. Altogether, we propose a model in which conformational transitions observed in PimA might reflect a molten globule state that confers to PimA, a higher affinity toward the dynamic and highly fluctuating lipid bilayer.
Asunto(s)
Proteínas Bacterianas/metabolismo , Manosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Mycobacterium smegmatis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dicroismo Circular , Escherichia coli/genética , Manosiltransferasas/química , Manosiltransferasas/genética , Proteínas de la Membrana/química , Modelos Moleculares , Fosfolípidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Secondary structure refolding is a key event in biology as it modulates the conformation of many proteins in the cell, generating functional or aberrant states. The crystal structures of mannosyltransferase PimA reveal an exceptional flexibility of the protein along the catalytic cycle, including ß-strand-to-α-helix and α-helix-to-ß-strand transitions. These structural changes modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane.
Asunto(s)
Proteínas Bacterianas/química , Membrana Celular/metabolismo , Glicosiltransferasas/metabolismo , Manosiltransferasas/química , Estructura Secundaria de Proteína , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Membrana Celular/enzimología , Cristalografía por Rayos X , Humanos , Manosiltransferasas/genética , Manosiltransferasas/aislamiento & purificación , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfolípidos/metabolismo , Estructura Secundaria de Proteína/genéticaRESUMEN
Histidine kinases (HKs) are dimeric receptors that participate in most adaptive responses to environmental changes in prokaryotes. Although it is well established that stimulus perception triggers autophosphorylation in many HKs, little is known on how the input signal propagates through the HAMP domain to control the transient interaction between the histidine-containing and ATP-binding domains during the catalytic reaction. Here we report crystal structures of the full cytoplasmic region of CpxA, a prototypical HK involved in Escherichia coli response to envelope stress. The structural ensemble, which includes the Michaelis complex, unveils HK activation as a highly dynamic process, in which HAMP modulates the segmental mobility of the central HK α-helices to promote a strong conformational and dynamical asymmetry that characterizes the kinase-active state. A mechanical model based on our structural and biochemical data provides insights into HAMP-mediated signal transduction, the autophosphorylation reaction mechanism, and the symmetry-dependent control of HK kinase/phosphatase functional states.
Asunto(s)
Adenosina Trifosfato/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Modelos Moleculares , Proteínas Quinasas/química , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Histidina/química , Histidina/metabolismo , Cinética , Movimiento (Física) , Fosforilación , Unión Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Termodinámica , Difracción de Rayos XRESUMEN
The histidine kinases belong to the family of two-component systems, which serves in bacteria to couple environmental stimuli to adaptive responses. Most of the histidine kinases are homodimers, in which the HAMP and DHp domains assemble into an elongated helical region flanked by two CA domains. Recently, X-ray crystallographic structures of the cytoplasmic region of the Escherichia coli histidine kinase CpxA were determined and a phosphotransferase-defective mutant, M228V, located in HAMP, was identified. In the present study, we recorded 1 µs molecular dynamics trajectories to compare the behavior of the WT and M228V protein dimers. The M228V modification locally induces the appearance of larger voids within HAMP as well as a perturbation of the number of voids within DHp, thus destabilizing the HAMP and DHp hydrophobic packing. In addition, a disruption of the stacking interaction between F403 located in the lid of the CA domain involved in the auto-phosphorylation and R296 located in the interacting DHp region, is more often observed in the presence of the M228V modification. Experimental modifications R296A and R296D of CpxA have been observed to reduce also the CpxA activity. These observations agree with the destabilization of the R296/F403 stacking, and could be the sign of the transmission of a conformational event taking place in HAMP to the auto-phosphorylation site of histidine kinase. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 670-682, 2016.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Mutación Missense , Proteínas Quinasas/química , Sustitución de Aminoácidos , Cristalografía por Rayos X , Estabilidad de Enzimas , Escherichia coli/química , Proteínas de Escherichia coli/genética , Dominios Proteicos , Proteínas Quinasas/genéticaRESUMEN
Signal transduction mediated by Ser/Thr phosphorylation in Mycobacterium tuberculosis has been intensively studied in the last years, as its genome harbors eleven genes coding for eukaryotic-like Ser/Thr kinases. Here we describe the crystal structure and the autophosphorylation sites of the catalytic domain of PknA, one of two protein kinases essential for pathogen's survival. The structure of the ligand-free kinase domain shows an auto-inhibited conformation similar to that observed in human Tyr kinases of the Src-family. These results reinforce the high conservation of structural hallmarks and regulation mechanisms between prokaryotic and eukaryotic protein kinases.
Asunto(s)
Proteínas Bacterianas/química , Mycobacterium tuberculosis/enzimología , Proteínas Serina-Treonina Quinasas/química , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Procesamiento Proteico-Postraduccional , Estructura Secundaria de ProteínaRESUMEN
The biosynthesis of membrane lipids is an essential pathway for virtually all bacteria. Despite its potential importance for the development of novel antibiotics, little is known about the underlying signaling mechanisms that allow bacteria to control their membrane lipid composition within narrow limits. Recent studies disclosed an elaborate feed-forward system that senses the levels of malonyl-CoA and modulates the transcription of genes that mediate fatty acid and phospholipid synthesis in many Gram-positive bacteria including several human pathogens. A key component of this network is FapR, a transcriptional regulator that binds malonyl-CoA, but whose mode of action remains enigmatic. We report here the crystal structures of FapR from Staphylococcus aureus (SaFapR) in three relevant states of its regulation cycle. The repressor-DNA complex reveals that the operator binds two SaFapR homodimers with different affinities, involving sequence-specific contacts from the helix-turn-helix motifs to the major and minor grooves of DNA. In contrast with the elongated conformation observed for the DNA-bound FapR homodimer, binding of malonyl-CoA stabilizes a different, more compact, quaternary arrangement of the repressor, in which the two DNA-binding domains are attached to either side of the central thioesterase-like domain, resulting in a non-productive overall conformation that precludes DNA binding. The structural transition between the DNA-bound and malonyl-CoA-bound states of SaFapR involves substantial changes and large (>30 Å) inter-domain movements; however, both conformational states can be populated by the ligand-free repressor species, as confirmed by the structure of SaFapR in two distinct crystal forms. Disruption of the ability of SaFapR to monitor malonyl-CoA compromises cell growth, revealing the essentiality of membrane lipid homeostasis for S. aureus survival and uncovering novel opportunities for the development of antibiotics against this major human pathogen.
Asunto(s)
Malonil Coenzima A/metabolismo , Lípidos de la Membrana/genética , Staphylococcus aureus/metabolismo , Factores de Transcripción/ultraestructura , Transcripción Genética , Antibacterianos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proliferación Celular , Cristalografía por Rayos X , Proteínas de Unión al ADN , Regulación Bacteriana de la Expresión Génica , Secuencias Hélice-Giro-Hélice/genética , Lípidos de la Membrana/biosíntesis , Conformación Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Infecciones Estafilocócicas , Staphylococcus aureus/genética , Factores de Transcripción/metabolismoRESUMEN
α-Ketoacid dehydrogenases are large multi-enzyme machineries that orchestrate the oxidative decarboxylation of α-ketoacids with the concomitant production of acyl-CoA and NADH. The first reaction, catalysed by α-ketoacid decarboxylases (E1 enzymes), needs a thiamine diphosphate cofactor and represents the overall rate-limiting step. Although the catalytic cycles of E1 from the pyruvate dehydrogenase (E1p) and branched-chain α-ketoacid dehydrogenase (E1b) complexes have been elucidated, little structural information is available on E1o, the first component of the α-ketoglutarate dehydrogenase complex, despite the central role of this complex at the branching point between the TCA (tricarboxylic acid) cycle and glutamate metabolism. In the present study, we provide structural evidence that MsKGD, the E1o (α-ketoglutarate decarboxylase) from Mycobacterium smegmatis, shows two conformations of the post-decarboxylation intermediate, each one associated with a distinct enzyme state. We also provide an overall picture of the catalytic cycle, reconstructed by either crystallographic snapshots or modelling. The results of the present study show that the conformational change leading the enzyme from the initial (early) to the late state, although not required for decarboxylation, plays an essential role in catalysis and possibly in the regulation of mycobacterial E1o.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carboxiliasas/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/enzimología , Procesamiento Proteico-Postraduccional , Replegamiento Proteico , Adipatos/química , Adipatos/metabolismo , Transferasas de Aldehído-Cetona/química , Transferasas de Aldehído-Cetona/genética , Transferasas de Aldehído-Cetona/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Carboxiliasas/química , Carboxiliasas/genética , Dominio Catalítico , Descarboxilación , Complejo Cetoglutarato Deshidrogenasa/química , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Conformación Molecular , Simulación del Acoplamiento Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Among the few proteins shown to be secreted by the Tat system in Mycobacterium tuberculosis, Rv2525c is of particular interest, since its gene is conserved in the minimal genome of Mycobacterium leprae. Previous evidence linked this protein to cell wall metabolism and sensitivity to ß-lactams. We describe here the crystal structure of Rv2525c that shows a TIM barrel-like fold characteristic of glycoside hydrolases of the GH25 family, which includes prokaryotic and phage-encoded peptidoglycan hydrolases. Structural comparison with other members of this family combined with substrate docking suggest that, although the 'neighbouring group' catalytic mechanism proposed for this family still appears as the most plausible, the identity of residues involved in catalysis in GH25 hydrolases might need to be revised.
Asunto(s)
Proteínas Bacterianas/metabolismo , Productos del Gen tat/metabolismo , Mycobacterium tuberculosis/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/química , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Secuencia de Aminoácidos , Catálisis , Dominio Catalítico , Pared Celular/metabolismo , Cristalografía por Rayos X/métodos , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Membrane-associated GT-B glycosyltransferases (GTs) comprise a large family of enzymes that catalyze the transfer of a sugar moiety from nucleotide-sugar donors to a wide range of membrane-associated acceptor substrates, mostly in the form of lipids and proteins. As a consequence, they generate a significant and diverse amount of glycoconjugates in biological membranes, which are particularly important in cell-cell, cell-matrix and host-pathogen recognition events. Membrane-associated GT-B enzymes display two "Rossmann-fold" domains separated by a deep cleft that includes the catalytic center. They associate permanently or temporarily to the phospholipid bilayer by a combination of hydrophobic and electrostatic interactions. They have the remarkable property to access both hydrophobic and hydrophilic substrates that reside within chemically distinct environments catalyzing their enzymatic transformations in an efficient manner. Here, we discuss the considerable progress that has been made in recent years in understanding the molecular mechanism that governs substrate and membrane recognition, and the impact of the conformational transitions undergone by these GTs during the catalytic cycle.
Asunto(s)
Glicosiltransferasas/química , Glicosiltransferasas/fisiología , Animales , Catálisis , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Relación Estructura-ActividadRESUMEN
Alpha-ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α-ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non-pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient-dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Genes Bacterianos , Ácidos Cetoglutáricos/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Línea Celular Tumoral , Ciclo del Ácido Cítrico , Regulación Bacteriana de la Expresión Génica , Ácido Glutámico/metabolismo , Humanos , Macrófagos/microbiología , Mutagénesis Sitio-Dirigida , Mycobacterium smegmatis/metabolismo , Fenotipo , Fosforilación , Proteínas Recombinantes/metabolismoRESUMEN
MoeA, or gephyrin in higher eukaryotes, is crucial for molybdenum cofactor biosynthesis required in redox reactions. Gephyrin is a moonlighting protein also involved in postsynaptic receptor clustering, a feature thought to be a recent evolutionary trait. We showed previously that a repurposed copy of MoeA (Glp) is involved in bacterial cell division. To investigate how MoeA acquired multifunctionality, we used phylogenetic inference and protein structure analyses to understand the diversity and evolutionary history of MoeA. Glp-expressing Bacteria have at least two copies of the gene, and our analysis suggests that Glp has lost its enzymatic role. In Archaea we identified an ancestral duplication where one of the paralogs might bind tungsten instead of molybdenum. In eukaryotes, the acquisition of the moonlighting activity of gephyrin comprised three major events: first, MoeA was obtained from Bacteria by early eukaryotes, second, MogA was fused to the N-terminus of MoeA in the ancestor of opisthokonts, and finally, it acquired the function of anchoring GlyR receptors in neurons. Our results support the functional versatility and adaptive nature of the MoeA scaffold, which has been repurposed independently both in eukaryotes and bacteria to carry out analogous functions in network organization at the cell membrane.