RESUMEN
ADP-ribosylation is a prominent and versatile post-translational modification, which regulates a diverse set of cellular processes. Poly-ADP-ribose (PAR) is synthesised by the poly-ADP-ribosyltransferases PARP1, PARP2, tankyrase (TNKS), and tankyrase 2 (TNKS2), all of which are linked to human disease. PARP1/2 inhibitors have entered the clinic to target cancers with deficiencies in DNA damage repair. Conversely, tankyrase inhibitors have continued to face obstacles on their way to clinical use, largely owing to our limited knowledge of their molecular impacts on tankyrase and effector pathways, and linked concerns around their tolerability. Whilst detailed structure-function studies have revealed a comprehensive picture of PARP1/2 regulation, our mechanistic understanding of the tankyrases lags behind, and thereby our appreciation of the molecular consequences of tankyrase inhibition. Despite large differences in their architecture and cellular contexts, recent structure-function work has revealed striking parallels in the regulatory principles that govern these enzymes. This includes low basal activity, activation by intra- or inter-molecular assembly, negative feedback regulation by auto-PARylation, and allosteric communication. Here we compare these poly-ADP-ribosyltransferases and point towards emerging parallels and open questions, whose pursuit will inform future drug development efforts.
Asunto(s)
Poli(ADP-Ribosa) Polimerasa-1 , Tanquirasas , Tanquirasas/metabolismo , Tanquirasas/antagonistas & inhibidores , Tanquirasas/genética , Tanquirasas/química , Humanos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/genética , Animales , Procesamiento Proteico-Postraduccional , Reparación del ADN , ADP-Ribosilación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli ADP Ribosilación/genéticaRESUMEN
Cdc14 phosphatases are related structurally and mechanistically to protein tyrosine phosphatases (PTPs) but evolved a unique specificity for phosphoSer-Pro-X-Lys/Arg sites primarily deposited by cyclin-dependent kinases. This specialization is widely conserved in eukaryotes. The evolutionary reconfiguration of the Cdc14 active site to selectively accommodate phosphoSer-Pro likely required modification to the canonical PTP catalytic cycle. While studying Saccharomyces cerevisiae Cdc14, we discovered a short sequence in the disordered C terminus, distal to the catalytic domain, which mimics an optimal substrate. Kinetic analyses demonstrated this pseudosubstrate binds the active site and strongly stimulates rate-limiting phosphoenzyme hydrolysis, and we named it "substrate-like catalytic enhancer" (SLiCE). The SLiCE motif is found in all Dikarya fungal Cdc14 orthologs and contains an invariant glutamine, which we propose is positioned via substrate-like contacts to assist orientation of the hydrolytic water, similar to a conserved active site glutamine in other PTPs that Cdc14 lacks. AlphaFold2 predictions revealed vertebrate Cdc14 orthologs contain a conserved C-terminal alpha helix bound to the active site. Although apparently unrelated to the fungal sequence, this motif also makes substrate-like contacts and has an invariant glutamine in the catalytic pocket. Altering these residues in human Cdc14A and Cdc14B demonstrated that it functions by the same mechanism as the fungal motif. However, the fungal and vertebrate SLiCE motifs were not functionally interchangeable, illuminating potential active site differences during catalysis. Finally, we show that the fungal SLiCE motif is a target for phosphoregulation of Cdc14 activity. Our study uncovered evolution of an unusual stimulatory pseudosubstrate motif in Cdc14 phosphatases.
RESUMEN
A host of metabolic enzymes reversibly self-assemble to form membrane-less, intracellular filaments under normal physiological conditions and in response to stress. Often, these enzymes reside at metabolic control points, suggesting that filament formation affords an additional regulatory mechanism. Examples include cytidine-5'-triphosphate (CTP) synthase (CTPS), which catalyzes the rate-limiting step for the de novo biosynthesis of CTP; inosine-5'-monophosphate dehydrogenase (IMPDH), which controls biosynthetic access to guanosine-5'-triphosphate (GTP); and ∆1-pyrroline-5-carboxylate (P5C) synthase (P5CS) that catalyzes the formation of P5C, which links the Krebs cycle, urea cycle, and proline metabolism. Intriguingly, CTPS can exist in co-assemblies with IMPDH or P5CS. Since GTP is an allosteric activator of CTPS, the association of CTPS and IMPDH filaments accords with the need to coordinate pyrimidine and purine biosynthesis. Herein, a hypothesis is presented furnishing a biochemical connection underlying co-assembly of CTPS and P5CS filaments - potent inhibition of CTPS by glutamate γ-semialdehyde, the open-chain form of P5C.
Asunto(s)
Ligasas de Carbono-Nitrógeno , IMP Deshidrogenasa , Animales , Humanos , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Citidina Trifosfato/metabolismo , Guanosina Trifosfato/metabolismo , IMP Deshidrogenasa/metabolismoRESUMEN
Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-EM. The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg2+ and define how the Mg2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg2+ in the SgrAI active site as a direct result of filamentation induced conformational changes.
Asunto(s)
División del ADN , Desoxirribonucleasas de Localización Especificada Tipo II , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/química , Dominio Catalítico , ADN/metabolismo , ADN/química , Microscopía por Crioelectrón , Magnesio/metabolismo , Magnesio/química , Cationes Bivalentes/metabolismo , Modelos MolecularesRESUMEN
Regulatory cystathionine ß-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.
Asunto(s)
Cistationina betasintasa , Cistationina betasintasa/metabolismo , Cistationina betasintasa/química , Cistationina betasintasa/genética , Mutación , Unión Proteica , Mutagénesis Sitio-Dirigida , Nucleótidos de Adenina/metabolismo , Nucleótidos de Adenina/química , Dominios Proteicos , Pirofosfatasas/metabolismo , Pirofosfatasas/química , Pirofosfatasas/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Pirofosfatasa Inorgánica/metabolismo , Pirofosfatasa Inorgánica/química , Pirofosfatasa Inorgánica/genética , Modelos Moleculares , Sitios de UniónRESUMEN
Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.
Asunto(s)
Oxidorreductasas , Vibrio , Oxidorreductasas/metabolismo , NAD/metabolismo , Cinamatos , Oxidación-Reducción , Vibrio/genética , Vibrio/metabolismo , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , NADH Deshidrogenasa/metabolismo , Flavinas/química , Transferasas , Flavina-Adenina Dinucleótido/metabolismoRESUMEN
The modern global trend toward sustainable processes that meet the requirements of "green chemistry" provides new opportunities for the broad application of highly active, selective, and specific enzymatic reactions. However, the effective application of enzymes in industrial processes requires the development of systems for the remote regulation of their activity triggered by external physical stimuli, one of which is a low-frequency magnetic field (LFMF). Magnetic nanoparticles (MNPs) transform the energy of an LFMF into mechanical forces and deformations applied to enzyme molecules on the surfaces of MNPs. Here, we demonstrate the up- and down-regulation of two biotechnologically important enzymes, yeast alcohol dehydrogenase (YADH) and soybean formate dehydrogenase (FDH), in aggregates with gold-covered magnetic nanoparticles (GCMNPs) triggered by an LFMF. Two types of aggregates, "dimeric" (with the enzyme attached to several GCMNPs simultaneously), with YADH or FDH, and "monomeric" (the enzyme attached to only one GCMNP), with FDH, were synthesized. Depending on the aggregate type ("dimeric" or "monomeric"), LFMF treatment led to a decrease (down-regulation) or an increase (up-regulation) in enzyme activity. For "dimeric" aggregates, we observed 67 ± 9% and 47 ± 7% decreases in enzyme activity under LFMF exposure for YADH and FDH, respectively. Moreover, in the case of YADH, varying the enzyme or the cross-linking agent concentration led to different magnitudes of the LFMF effect, which was more significant at lower enzyme and higher cross-linking agent concentrations. Different responses to LFMF exposure depending on cofactor presence were also demonstrated. This effect might result from a varying cofactor binding efficiency to enzymes. For the "monomeric" aggregates with FDH, the LFMF treatment caused a significant increase in enzyme activity; the magnitude of this effect depended on the cofactor type: we observed up to 40% enzyme up-regulation in the case of NADP+, while almost no effect was observed in the case of NAD+.
RESUMEN
De novo purine nucleotide biosynthesis (DNPNB) consists of sequential reactions that are majorly conserved in living organisms. Several regulation events take place to maintain physiological concentrations of adenylate and guanylate nucleotides in cells and to fine-tune the production of purine nucleotides in response to changing cellular demands. Recent years have seen a renewed interest in the DNPNB enzymes, with some being highlighted as promising targets for therapeutic molecules. Herein, a review of two newly revealed modes of regulation of the DNPNB pathway has been carried out: i) the unprecedent allosteric regulation of one of the limiting enzymes of the pathway named inosine 5'-monophosphate dehydrogenase (IMPDH), and ii) the supramolecular assembly of DNPNB enzymes. Moreover, recent advances that revealed the therapeutic potential of DNPNB enzymes in bacteria could open the road for the pharmacological development of novel antibiotics.
RESUMEN
Nucleotide biosynthesis encompasses both de novo and salvage synthesis pathways, each characterized by significant material and procedural distinctions. Despite these differences, cells with elevated nucleotide demands exhibit a preference for the more intricate de novo synthesis pathway, intricately linked to modes of enzyme regulation. In this study, we primarily scrutinize the biological importance of a conserved yet promising mode of enzyme regulation in nucleotide metabolism-cytoophidia. Cytoophidia, comprising cytidine triphosphate synthase or inosine monophosphate dehydrogenase, is explored across diverse biological models, including yeasts, Drosophila, mice, and human cancer cell lines. Additionally, we delineate potential biomedical applications of cytoophidia. As our understanding of cytoophidia deepens, the roles of enzyme compartmentalization and polymerization in various biochemical processes will unveil, promising profound impacts on both research and the treatment of metabolism-related diseases.
Asunto(s)
Fenómenos Bioquímicos , Drosophila , Humanos , Animales , Ratones , Línea Celular , Modelos Biológicos , NucleótidosRESUMEN
Human copper transporters ATP7B and ATP7A deliver copper to biosynthetic pathways and maintain copper homeostasis in the cell. These enzymes combine several challenges for structural biology because they are large low abundance membrane proteins with many highly mobile domains and long disordered loops. No method has yet succeeded in solving the structure of the complete fully functional protein. Still, X-ray crystallography, Cryo-EM and NMR helped to piece together a structure based model of the enzyme activity and regulation by copper. We review the structures of ATP7B and ATP7A with an emphasis on the mechanistic insights into the unique aspects of the transport function and regulation of the human copper ATPases that have emerged from more than twenty years of research.
Asunto(s)
Proteínas de Transporte de Catión , Cobre , Humanos , ATPasas Transportadoras de Cobre/genética , ATPasas Transportadoras de Cobre/metabolismo , Cobre/química , Proteínas de Transporte de Catión/metabolismo , HomeostasisRESUMEN
Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.
Asunto(s)
Malaria Falciparum , Plasmodium , Animales , Humanos , Subtilisina/metabolismo , Merozoítos/fisiología , Dimerización , Proteínas Protozoarias/metabolismo , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Eritrocitos/parasitología , Concentración de Iones de HidrógenoRESUMEN
IMPORTANCE: A peptidoglycan cell wall is an essential component of almost all bacterial cell envelopes, which determines cell shape and prevents osmotic rupture. Antibiotics that interfere with peptidoglycan synthesis have been one of the most important treatments for bacterial infections. Peptidoglycan must also be hydrolyzed to incorporate new material for cell growth and division and to help accommodate important envelope-spanning systems. However, the enzymes that hydrolyze peptidoglycan must be carefully controlled to prevent autolysis. Exactly how this control is achieved is poorly understood in most cases but is a highly active area of current research. Identifying hydrolase control mechanisms has the potential to provide new targets for therapeutic intervention. The work here reports the important discovery of a novel inhibitor/anti-inhibitor system that controls the activity of a cell wall hydrolase in the human pathogen Pseudomonas aeruginosa, which also affects resistance to an antibiotic used in the clinic.
RESUMEN
We previously demonstrated that Aedes aegypti pyruvate kinase (AaPK) plays a key role in the regulation of both carbon and nitrogen metabolism in mosquitoes. To further elucidate whether AaPK can be post-translationally regulated by Ae. aegypti sirtuin 2 (AaSirt2), an NAD+-dependent deacetylase that catalyzes the removal of acetyl groups from acetylated lysine residues, we conducted a series of analysis in non-starved and starved female mosquitoes. Transcriptional and protein profiles of AaSirt2, analyzed by qPCR and western blots, indicated that the AaSirt2 is differentially modulated in response to sugar or blood feeding in mosquito tissues dissected at different times during the first gonotrophic cycle. We also found that AaSirt2 is localized in both cytosolic and mitochondrial cellular compartments of fat body and thorax. Multiple lysine-acetylated proteins were detected by western blotting in both cellular compartments. Furthermore, western blotting of immunoprecipitated proteins provided evidence that AaPK is lysine-acetylated and bound with AaSirt2 in the cytosolic fractions of fat body and thorax from non-starved and starved females. In correlation with these results, we also discovered that RNAi-mediated knockdown of AaSirt2 in the fat body of starved females significantly decreased AaPK protein abundance. Notably, survivorship of AaSirt2-deficient females maintained under four different nutritional regimens was not significantly affected. Taken together, our data reveal that AaPK is post-translationally regulated by AaSirt2.
Asunto(s)
Aedes , Femenino , Animales , Aedes/metabolismo , Piruvato Quinasa/metabolismo , Sirtuina 2/metabolismo , Lisina/metabolismo , Interferencia de ARNRESUMEN
Pteridine reductase 1 (PTR1) is a folate and pterin pathway enzyme unique for pathogenic trypanosomatids. As a validated drug target, PTR1 has been the focus of recent research efforts aimed at finding more effective treatments against human parasitic diseases such as leishmaniasis or sleeping sickness. Previous PTR1-centered structural studies highlighted the enzyme characteristics, such as flexible regions around the active site, highly conserved structural waters, and species-specific differences in pocket properties and dynamics, which likely impacts the binding of natural substrates and inhibitors. Furthermore, several aspects of the PTR1 function, such as the substrate inhibition phenomenon and the level of ligand binding cooperativity in the enzyme homotetramer, likely related to the global enzyme dynamics, are poorly known at the molecular level. We postulate that future drug design efforts could greatly benefit from a better understanding of these phenomena through studying both the local and global PTR1 dynamics. This review highlights the key aspects of the PTR1 structure and dynamics relevant to structure-based drug design that could be effectively investigated by modeling approaches. Particular emphasis is given to the perspective of molecular dynamics, what has been accomplished in this area to date, and how modeling could impact the PTR1-targeted drug design in the future.
Asunto(s)
Diseño de Fármacos , Oxidorreductasas , Humanos , Oxidorreductasas/química , Simulación de Dinámica Molecular , Inhibidores Enzimáticos/farmacologíaRESUMEN
Data on protein post-translational modifications (PTMs) increased exponentially in the last years due to the refinement of mass spectrometry techniques and the development of databases to store and share datasets. Nevertheless, these data per se do not create comprehensive biochemical knowledge. Complementary studies on protein biochemistry are necessary to fully understand the function of these PTMs at the molecular level and beyond, for example, designing rational metabolic engineering strategies to improve crops. Phosphoenolpyruvate carboxykinases (PEPCKs) are critical enzymes for plant metabolism with diverse roles in plant development and growth. Multiple lines of evidence showed the complex regulation of PEPCKs, including PTMs. Herein, we present PEPCKs as an example of the integration of combined mechanisms modulating enzyme activity and metabolic pathways. PEPCK studies strongly advanced after the production of the recombinant enzyme and the establishment of standardized biochemical assays. Finally, we discuss emerging open questions for future research and the challenges in integrating all available data into functional biochemical models.
RESUMEN
Half-of-sites reactivity in many homodimeric and homotetrameric metalloenzymes has been known for half a century, yet its benefit remains poorly understood. A recently reported cryo-electron microscopy structure has given some clues on the less optimized reactivity of Escherichia coli ribonucleotide reductase with an asymmetric association of α2ß2 subunits during catalysis. Moreover, nonequivalence of enzyme active sites has been reported in many other enzymes, possibly as a means of regulation. They are often induced by substrate binding or caused by a critical component introduced from a neighboring subunit in response to substrate loadings, such as in prostaglandin endoperoxide H synthase, cytidine triphosphate synthase, glyoxalase, tryptophan dioxygenase, and several decarboxylases or dehydrogenases. Overall, half-of-sites reactivity is likely not an act of wasting resources but rather a method devised in nature to accommodate catalytic or regulatory needs.
Asunto(s)
Metaloproteínas , Microscopía por Crioelectrón , Metaloproteínas/química , Dominio Catalítico , Escherichia coli , Catálisis , Sitios de UniónRESUMEN
The wood frog, Rana sylvatica endures whole body freezing for weeks/months while overwintering at subzero temperatures. Survival of long-term freezing requires not only cryoprotectants but also strong metabolic rate depression (MRD) and reorganization of essential processes in order to maintain a balance between ATP-producing and ATP-consuming processes. Citrate synthase (CS) (E.C. 2.3.3.1) is an important irreversible enzyme of the tricarboxylic acid (TCA) cycle and forms a crucial checkpoint for many metabolic processes. Present study investigated the regulation of CS from wood frog liver during freezing. CS was purified to homogeneity by a two-step chromatographic process. Kinetic and regulatory parameters of the enzyme were investigated and, notably, demonstrated a significant decrease in the Vmax of the purified form of CS from frozen frogs as compared to controls when assayed at both 22 °C and 5 °C. This was further supported by a decrease in the maximum activity of CS from liver of frozen frogs. Immunoblotting also showed changes in posttranslational modifications with a significant decrease in threonine phosphorylation (by 49 %) for CS from frozen frogs. Taken together, these results suggest that CS is suppressed and TCA flux is inhibited during freezing, likely to support MRD survival of harsh winters.
Asunto(s)
Hígado , Ranidae , Animales , Congelación , Citrato (si)-Sintasa/metabolismo , Ranidae/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.
Asunto(s)
Carboxiliasas , Malaria , Fosfolípidos , Plasmodium , Secuencias de Aminoácidos , Calcio/metabolismo , Calcio/farmacología , Carboxiliasas/antagonistas & inhibidores , Carboxiliasas/química , Carboxiliasas/metabolismo , Precursores Enzimáticos/metabolismo , Liposomas , Ácidos Fosfatidicos/metabolismo , Ácidos Fosfatidicos/farmacología , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/farmacología , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/farmacología , Fosfatidilgliceroles/metabolismo , Fosfatidilgliceroles/farmacología , Fosfatidilinositoles/metabolismo , Fosfatidilinositoles/farmacología , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacología , Fosfolípidos/química , Fosfolípidos/metabolismo , Fosfolípidos/farmacología , Unión Proteica , Malaria/parasitología , Proteolisis/efectos de los fármacos , Resonancia por Plasmón de Superficie , Plasmodium/enzimologíaRESUMEN
Microbial co-culture simulates the natural ecosystem through the combination of artificial microbes. This approach has been widely applied in the study of activating silent genes to reveal novel secondary metabolites. However, there are still challenges in determining the biosynthetic pathways. In this study, the effects of microbial co-culture on the morphology of the microbes were verified by the morphological observation. Subsequently, through the strategy combining substrate feeding, stable isotope labeling, and gene expression analysis, the biosynthetic pathways of five benzoic acid derivatives N1-N4 and N7 were demonstrated: the secondary metabolite 10-deoxygerfelin of A. sydowii acted as an inducer to induce B. subtilis to produce benzoic acid, which was further converted into 3-OH-benzoic acid by A. sydowii. Subsequently, A. sydowii used 3-OH-benzoic acid as the substrate to synthesize the new compound N2, and then N1, N3, N4, and N7 were biosynthesized upon the upregulation of hydrolase, hydroxylase, and acyltransferase during co-culture. The plate zone analysis suggested that the biosynthesis of the newly induced compounds N1-N4 was mainly attributed to A. sydowii, and both A. sydowii and B. subtilis were indispensable for the biosynthesis of N7. This study provides an important basis for a better understanding of the interactions among microorganisms, providing new ideas for studying the biosynthetic pathways of the newly induced secondary metabolites in co-culture.
Asunto(s)
Bacillus subtilis , Ecosistema , Bacillus subtilis/genética , Técnicas de Cocultivo , Ácido BenzoicoRESUMEN
The wood frog (Rana sylvatica) undergoes physiological and metabolic changes to withstand subzero temperatures and whole body freezing during the winter months. Along with metabolic rate depression, high concentrations of glucose are produced as a cryoprotectant by liver and distributed to all other tissues. Pyruvate kinase (PK; EC:2.7.1.40), the final enzyme of glycolysis, plays an important role in the modulation of glucose metabolism and, therefore, overall metabolic regulation. The present study investigated the functional and kinetic properties of purified PK from liver of control (5 °C acclimated) and frozen (-2.5 °C for 24 h) wood frogs. Liver PK was purified to homogeneity by a two-step chromatographic process, followed by analysis of enzyme properties. A significant decrease in the affinity of PK for its substrates, phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) at 22 °C and 5 °C was noted in liver from frozen frogs, as compared with controls. Immunoblotting also revealed freeze-responsive changes in posttranslational modifications with a significant increase in serine and threonine phosphorylation by 1.46-fold and 1.73- fold for PK from frozen frogs as compared with controls. Furthermore, a test of thermal stability showed that PK from liver of frozen wood frogs showed greater stability as compared with PK from control animals. Taken together, these results suggest that PK is negatively regulated, and glycolysis is suppressed, during freezing. This response acts as an important survival strategy for maintaining continuously elevated levels of cryoprotectant in frogs while they remain in a hypometabolic frozen state.