RESUMO
BACKGROUND: Triokinase and FMN cyclase (TKFC) is a bifunctional enzyme involved in fructose metabolism. Triokinase catalyses the phosphorylation of fructose-derived glyceraldehyde (GA) and exogenous dihydroxyacetone (DHA), while FMN cyclase generates cyclic FMN. TKFC regulates the antiviral immune response by interacting with IFIH1 (MDA5). Previously reported pathogenic variants in TKFC are associated with either a multisystemic disease or isolated hypotrichosis with loose anagen hairs. METHODS: Whole-exome sequencing identified a homozygous novel variant in TKFC (c.1624G>A; p.Gly542Arg) in an individual with a complex primary immunodeficiency disorder. The variant was characterised using enzymatic assays and yeast studies of mutant recombinant proteins. RESULTS: The individual presented with chronic active Epstein-Barr virus disease and multiple bacterial and viral infections. Clinical investigations revealed hypogammaglobulinaemia, near absent natural killer cells and decreased memory B cells. Enzymatic assays showed that this variant displayed defective DHA and GA kinase activity while maintaining FMN cyclase activity. An allogenic bone marrow transplantation corrected the patient's immunodeficiency. CONCLUSION: Our report suggests that TKFC may have a role in the immunological system. The pathological features associated with this variant are possibly linked with DHA/GA kinase inactivation through a yet an unknown mechanism. This report thus adds a possible new pathway of immunometabolism to explore further.
RESUMO
The cpdB gene is pro-virulent in avian pathogenic Escherichia coli and in Salmonella enterica, where it encodes a periplasmic protein named CpdB. It is structurally related to cell wall-anchored proteins, CdnP and SntA, encoded by the also pro-virulent cdnP and sntA genes of Streptococcus agalactiae and Streptococcus suis, respectively. CdnP and SntA effects are due to extrabacterial hydrolysis of cyclic-di-AMP, and to complement action interference. The mechanism of CpdB pro-virulence is unknown, although the protein from non-pathogenic E. coli hydrolyzes cyclic dinucleotides. Considering that the pro-virulence of streptococcal CpdB-like proteins is mediated by c-di-AMP hydrolysis, S. enterica CpdB activity was tested as a phosphohydrolase of 3'-nucleotides, 2',3'-cyclic mononucleotides, linear and cyclic dinucleotides, and cyclic tetra- and hexanucleotides. The results help to understand cpdB pro-virulence in S. enterica and are compared with E. coli CpdB and S. suis SntA, including the activity of the latter on cyclic-tetra- and hexanucleotides reported here for the first time. On the other hand, since CpdB-like proteins are relevant to host-pathogen interactions, the presence of cpdB-like genes was probed in eubacterial taxa by TblastN analysis. The non-homogeneous genomic distribution revealed taxa with cpdB-like genes present or absent, identifying eubacteria and plasmids where they can be relevant.
Assuntos
Proteínas de Escherichia coli , Salmonella enterica , Streptococcus suis , Escherichia coli/metabolismo , Salmonella enterica/metabolismo , Streptococcus suis/metabolismo , Virulência , AMP Cíclico , Genômica , Proteínas de Escherichia coli/metabolismo , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/genéticaRESUMO
CpdB is a 3'-nucleotidase/2'3'-cyclic nucleotide phosphodiesterase, active also with reasonable efficiency on cyclic dinucleotides like c-di-AMP (3',5'-cyclic diadenosine monophosphate) and c-di-GMP (3',5'-cyclic diadenosine monophosphate). These are regulators of bacterial physiology, but are also pathogen-associated molecular patterns recognized by STING to induce IFN-ß response in infected hosts. The cpdB gene of Gram-negative and its homologs of gram-positive bacteria are virulence factors. Their protein products are extracytoplasmic enzymes (either periplasmic or cell-wall anchored) and can hydrolyze extracellular cyclic dinucleotides, thus reducing the innate immune responses of infected hosts. This makes CpdB(-like) enzymes potential targets for novel therapeutic strategies in infectious diseases, bringing about the necessity to gain insight into the molecular bases of their catalytic behavior. We have dissected the two-domain structure of Escherichia coli CpdB to study the role of its N-terminal and C-terminal domains (CpdB_Ndom and CpdB_Cdom). The specificity, kinetics and inhibitor sensitivity of point mutants of CpdB, and truncated proteins CpdB_Ndom and CpdB_Cdom were investigated. CpdB_Ndom contains the catalytic site, is inhibited by phosphate but not by adenosine, while CpdB_Cdom is inactive but contains a substrate-binding site that determines substrate specificity and adenosine inhibition of CpdB. Among CpdB substrates, 3'-AMP, cyclic dinucleotides and linear dinucleotides are strongly dependent on the CpdB_Cdom binding site for activity, as the isolated CpdB_Ndom showed much-diminished activity on them. In contrast, 2',3'-cyclic mononucleotides and bis-4-nitrophenylphosphate were actively hydrolyzed by CpdB_Ndom, indicating that they are rather independent of the CpdB_Cdom binding site.
Assuntos
2',3'-Nucleotídeo Cíclico Fosfodiesterases/química , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Adenosina/metabolismo , Biocatálise , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fosfatos/metabolismo , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/genética , Sítios de Ligação , Domínio Catalítico , Proteínas de Escherichia coli/genética , Histidina/metabolismo , Hidrólise , Cinética , Modelos Moleculares , Mutação Puntual/genética , Domínios Proteicos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The 5'-nucleotidase UshA and the 3'-nucleotidase CpdB from Escherichia coli are broad-specificity phosphohydrolases with similar two-domain structures. Their N-terminal domains (UshA_Ndom and CpdB_Ndom) contain the catalytic site, and their C-terminal domains (UshA_Cdom and CpdB_Cdom) contain a substrate-binding site responsible for specificity. Both enzymes show only partial overlap in their substrate specificities. So, it was decided to investigate the catalytic behavior of chimeras bearing the UshA catalytic domain and the CpdB specificity domain, or vice versa. UshA_Ndom-CpdB_Cdom and CpdB_Ndom-UshA_Cdom were constructed and tested on substrates specific to UshA (5'-AMP, CDP-choline, UDP-glucose) or to CpdB (3'-AMP), as well as on 2',3'-cAMP and on the common phosphodiester substrate bis-4-NPP (bis-4-nitrophenylphosphate). The chimeras did show neither 5'-nucleotidase nor 3'-nucleotidase activity. When compared to UshA, UshA_Ndom-CpdB_Cdom conserved high activity on bis-4-NPP, some on CDP-choline and UDP-glucose, and displayed activity on 2',3'-cAMP. When compared to CpdB, CpdB_Ndom-UshA_Cdom conserved phosphodiesterase activities on 2',3'-cAMP and bis-4-NPP, and gained activity on the phosphoanhydride CDP-choline. Therefore, the non-nucleotidase activities of UshA and CpdB are not fully dependent on the interplay between domains. The specificity domains may confer the chimeras some of the phosphodiester or phosphoanhydride selectivity displayed when associated with their native partners. Contrarily, the nucleotidase activity of UshA and CpdB depends strictly on the interplay between their native catalytic and specificity domains.
Assuntos
Nucleotidases/metabolismo , Sítios de Ligação , Catálise , Diester Fosfórico Hidrolases/metabolismo , Especificidade por SubstratoRESUMO
Human triokinase/flavin mononucleotide (FMN) cyclase (hTKFC) catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of D-glyceraldehyde and dihydroxyacetone (DHA), and the cyclizing splitting of flavin adenine dinucleotide (FAD). hTKFC structural models are dimers of identical subunits, each with two domains, K and L, with an L2-K1-K2-L1 arrangement. Two active sites lie between L2-K1 and K2-L1, where triose binds K and ATP binds L, although the resulting ATP-to-triose distance is too large (≈14 Å) for phosphoryl transfer. A 75-ns trajectory of molecular dynamics shows considerable, but transient, ATP-to-DHA approximations in the L2-K1 site (4.83 Å or 4.16 Å). To confirm the trend towards site closure, and its relationship to kinase activity, apo-hTKFC, hTKFC:2DHA:2ATP and hTKFC:2FAD models were submitted to normal mode analysis. The trajectory of hTKFC:2DHA:2ATP was extended up to 160 ns, and 120-ns trajectories of apo-hTKFC and hTKFC:2FAD were simulated. The three systems were comparatively analyzed for equal lengths (120 ns) following the principles of essential dynamics, and by estimating site closure by distance measurements. The full trajectory of hTKFC:2DHA:2ATP was searched for in-line orientations and short distances of DHA hydroxymethyl oxygens to ATP γ-phosphorus. Full site closure was reached only in hTKFC:2DHA:2ATP, where conformations compatible with an associative phosphoryl transfer occurred in L2-K1 for significant trajectory time fractions.
Assuntos
Apoenzimas/genética , Simulação de Dinâmica Molecular , Fósforo-Oxigênio Liases/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Trifosfato de Adenosina/química , Apoenzimas/química , Sítios de Ligação , Catálise , Domínio Catalítico/genética , Di-Hidroxiacetona/química , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/genética , Flavina-Adenina Dinucleotídeo/química , Gliceraldeído/química , Humanos , Fósforo-Oxigênio Liases/genética , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Especificidade por SubstratoRESUMO
Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈ 14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4'-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr(112) (hydrogen bonding of ATP adenine to K in the closed active center), His(221) (covalent anchoring of dihydroxyacetone to K), Asp(401) and Asp(403) (metal coordination to L), and Asp(556) (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His(221) point mutant acted specifically as a cyclase without kinase activity.
Assuntos
Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Animais , Catálise , Domínio Catalítico , Dimerização , Flavina-Adenina Dinucleotídeo/química , Frutose/química , Gliceraldeído/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Especificidade por Substrato , SuínosRESUMO
UshA and CpdB are nucleotidases of the periplasm of several Gram-negative bacteria, while several Gram-positives contain cell wall-bound variants. UshA is a 5'-nucleotidase, a UDP-sugar hydrolase, and a CDP-alcohol hydrolase. CpdB acts as a 3'-nucleotidase and as a phosphodiesterase of 2',3'-cyclic nucleotides and 3',5'-linear and cyclic dinucleotides. Both proteins are pro-virulent for the pathogens producing them and facilitate escape from the innate immunity of the infected host. Recently, the genomic distribution of cpdB-like genes in Bacteria was found to be non-homogeneous among different taxa, and differences occur within single taxa, even at species level. Similitudes and differences between UshA-like and CpdB-like proteins prompted parallel analysis of their genomic distributions in Bacteria. The presence of ushA-like and cpdB-like genes was tested by TBlastN analysis using seven protein probes to query the NCBI Complete Genomes Database. It is concluded that the distribution of ushA-like genes, like that of cpdB-like genes, is non-homogeneous. There is a partial correlation between both gene kinds: in some taxa, both are present or absent, while in others, only one is present. The result is an extensive catalog of the genomic distribution of these genes at different levels, from phylum to species, constituting a starting point for research using other in silico or experimental approaches.
Assuntos
Bactérias , Genômica , Bactérias/genética , Parede Celular , Etanol , HidrolasesRESUMO
TKFC-encoded triokinase catalyses glyceraldehyde phosphorylation in fructose metabolism and favours lipogenesis in mice. In Tkfc knockouts or knockdowns, fructose oxidation predominates over lipogenesis. The highly prevalent human variant Ala185Thr-Triokinase/FMN cyclase (TKFC) has been reported to be 'null' for fructose metabolism, since Ala185-TKFC rescues the mouse TKFC-deficient phenotype, whereas Ala185Thr-TKFC does not. Such report implies that most humans would display a noncanonical fructose metabolism, but it ignores the well-characterized triokinase activity of Ala185Thr-TKFC. Here, earlier evidence is summarized, along with new evidence that both human variants are equally active in yeast. Therefore, future research on triokinase in the context of human fructose metabolism should consider that Ala185Thr-TKFC is not biochemically 'null'.
Assuntos
Fígado , Fosfotransferases (Aceptor do Grupo Álcool) , Animais , Frutose/metabolismo , Gliceraldeído/química , Gliceraldeído/metabolismo , Fígado/metabolismo , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Streptococcus suis and Streptococcus agalactiae evade the innate immune system of the infected host by mechanisms mediated by cell wall-anchored proteins: SntA and CdnP, respectively. The former has been reported to interfere with complement responses, and the latter dampens STING-dependent type-I interferon (IFN) response by hydrolysis of bacterial cyclic-di-AMP (c-di-AMP). Both proteins are homologous but, while CdnP has been studied as a phosphohydrolase, the enzyme activities of SntA have not been investigated. The core structure of SntA was expressed in Escherichia coli as a GST-tagged protein that, after affinity purification, was characterized as phosphohydrolase with a large series of substrates. This included 3'-nucleotides, 2',3'-cyclic nucleotides, cyclic and linear dinucleotides, and a variety of phosphoanhydride or phosphodiester compounds, most of them previously considered as substrates of E. coli CpdB, a periplasmic protein homologous to SntA and CdnP. Catalytic efficiency was determined for each SntA substrate, either by dividing parameters k cat /K M obtained from saturation curves or directly from initial rates at low substrate concentrations when saturation curves could not be obtained. SntA is concluded to act as phosphohydrolase on two groups of substrates with efficiencies higher or lower than ≈ 105 M-1 s-1 (average value of the enzyme universe). The group with k cat /K M ≥ 105 M-1 s-1 (good substrates) includes 3'-nucleotides, 2',3'-cyclic nucleotides, and linear and cyclic dinucleotides (notably c-di-AMP). Compounds showing efficiencies <104 M-1 s-1 are considered poor substrates. Compared with CpdB, SntA is more efficient with its good substrates and less efficient with its poor substrates; therefore, the specificity of SntA is more restrictive. The efficiency of the SntA activity on c-di-AMP is comparable with the activity of CdnP that dampens type-I IFN response, suggesting that this virulence mechanism is also functional in S. suis. SntA modeling revealed that Y530 and Y633 form a sandwich with the nitrogen base of nucleotidic ligands in the substrate-binding site. Mutants Y530A-SntA, Y633A-SntA, and Y530A+Y633A-SntA were obtained and kinetically characterized. For orientation toward the catalytic site, one tyrosine is enough, although this may depend on the substrate being attacked. On the other hand, both tyrosines are required for the efficient binding of good SntA substrates.
RESUMO
A novel enzyme, induced by choline, ethanolamine, glycine betaine or dimethylglycine, was released at low temperature and phosphate from Pseudomonas fluorescens (CECT 7229) suspensions at low cell densities. It is a CDP-ethanolamine pyrophosphatase/(dihexanoyl)glycerophosphoethanolamine phosphodiesterase (CGDEase) less active on choline derivatives, and inactive on long-chain phospholipids, CDP-glycerol and other NDP-X compounds. The reaction pattern was typical of phospholipase C (PLC), as either phosphoethanolamine or phosphocholine was produced. Peptide-mass analyses, gene cloning and expression provided a molecular identity for CGDEase. Bioinformatic studies assigned it to the PLC branch of the phospholipase C/acid phosphatase (PLC/APase) superfamily, revealed an irregular phylogenetic distribution of close CGDEase relatives, and suggested their genes are not in operons or conserved contexts. A theoretical CGDEase structure was supported by mutagenesis of two predicted active-site residues, which yielded essentially inactive mutants. Biological relevance is supported by comparisons with CGDEase relatives, induction by osmoprotectants (not by osmotic stress itself) and repression by micromolar phosphate. The low bacterial density requirement was related to phosphate liberation from lysed bacteria in denser populations, rather than to a classical quorum-sensing effect. The results fit better a CGDEase role in phosphate scavenging than in osmoprotection.
Assuntos
Regulação Enzimológica da Expressão Gênica , Fosfatos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pseudomonas fluorescens/enzimologia , Pirofosfatases/metabolismo , Domínio Catalítico , Cistina Difosfato/análogos & derivados , Cistina Difosfato/metabolismo , Etanolaminas/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Família Multigênica , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Pseudomonas fluorescens/química , Pseudomonas fluorescens/genética , Pirofosfatases/química , Pirofosfatases/genética , Especificidade por SubstratoRESUMO
Flavins, comprising flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin (RF, vitamin B(2)), play important roles in numerous redox reactions such as those taking place in the electron-transfer chains of mitochondria in all eukaryotes and of plastids in plants. A selective chemosensor for flavins would be useful not only in the investigation of metabolic processes but also in the diagnosis of diseases related to flavins; such a sensor is presently unavailable. Herein, we report the first bifunctional chemosensor (PTZ-DPA) for flavins. PTZ-DPA consists of bis(Zn(2+)-dipicolylamine) and phenothiazine. Bis(Zn(2+)-dipicolylamine) (referred to here as XyDPA) was found to be an excellent catalyst in the conversion of FAD into cyclic FMN (riboflavin 4',5'-cyclic phosphate, cFMN) under physiological conditions, even at pH 7.4 and 27 degrees C, with less than 1 mol % of substrate. Utilizing XyDPA's superior function as an artificial FMN cyclase and phenothiazine as an electron donor able to quench the fluorescence of an isoalloxazine ring, PTZ-DPA enabled selective fluorescent discrimination of flavins (FMN, FAD, and RF): FAD shows ON(+), FMN shows OFF(-), and RF shows NO(0) fluorescence changes upon the addition of PTZ-DPA. With this selective sensing property, PTZ-DPA is applicable to real-time fluorescent monitoring of riboflavin kinase (RF to FMN), alkaline phosphatase (FMN to RF), and FAD synthetase (FMN to FAD).
Assuntos
Materiais Biomiméticos/química , Técnicas de Química Analítica/métodos , Flavinas/análise , Fluorescência , Compostos Organometálicos/química , Fósforo-Oxigênio Liases/metabolismo , Aminas/química , Materiais Biomiméticos/metabolismo , Mononucleotídeo de Flavina/análise , Mononucleotídeo de Flavina/metabolismo , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Medições Luminescentes , Estrutura Molecular , Ácidos Picolínicos/química , Zinco/químicaRESUMO
ADPRibase-Mn (Mn2+-dependent ADP-ribose/CDP-alcohol pyrophosphatase) was earlier isolated from rat liver supernatants after separation from ADPRibase-I and ADPRibase-II (Mg2+-activated ADP-ribose pyrophosphatases devoid of CDP-alcohol pyrophosphatase activity). The last mentioned are putative Nudix hydrolases, whereas the molecular identity of ADPRibase-Mn is unknown. MALDI (matrix-assisted laser-desorption ionization) MS data from rat ADPRibase-Mn pointed to a hypothetical protein that was cloned and expressed and showed the expected specificity. It is encoded by the RGD1309906 rat gene, which so far has been annotated simply as 'hydrolase'. ADPRibase-Mn is not a Nudix hydrolase, but it shows the sequence and structural features typical of the metallophosphoesterase superfamily. It may constitute a protein family of its own, the members of which appear to be specific to vertebrates, plants and algae. ADP-ribose was successfully docked to a model of rat ADPRibase-Mn, revealing its putative active centre. Microarray data from the GEO (Gene Expression Omnibus) database indicated that the mouse gene 2310004I24Rik, an orthologue of RGD1309906, is preferentially expressed in immune cells. This was confirmed by Northern-blot and activity assay of ADPRibase-Mn in rat tissues. A possible role of ADPRibase-Mn in immune cell signalling is suggested by the second-messenger role of ADP-ribose, which activates TRPM2 (transient receptor potential melastatin channel-2) ion channels as a mediator of oxidative/nitrosative stress, and by the signalling function assigned to many of the microarray profile neighbours of 2310004I24Rik. Furthermore, the influence of ADPRibase-Mn on the CDP-choline or CDP-ethanolamine pathways of phospholipid biosynthesis cannot be discounted.
Assuntos
Pirofosfatases/química , Pirofosfatases/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cistina Difosfato/análogos & derivados , Cistina Difosfato/metabolismo , Citidina Difosfato Colina/metabolismo , Etanolaminas/metabolismo , Feminino , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Fígado/enzimologia , Tecido Linfoide/metabolismo , Manganês/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Açúcares de Nucleosídeo Difosfato/metabolismo , Pirofosfatases/genética , Ratos , Ratos Wistar , Proteínas Recombinantes , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
Nucleoside 5'-diphosphate-X hydrolases are interesting enzymes to study due to their varied activities and structure-function relationships and the roles they play in the disposal, assimilation, and modulation of the effects of their substrates. Few of these enzymes with a preference for CDP-alcohols are known. In Yersinia intermedia suspensions prepared from cultures on Columbia agar with 5% sheep blood, we found a CDP-alcohol hydrolase liberated to Triton X-100-containing medium. Growth at 25 degrees C was deemed optimum in terms of the enzyme-activity yield. The purified enzyme also displayed 5'-nucleotidase, UDP-sugar hydrolase, and dinucleoside-polyphosphate hydrolase activities. It was identified as the protein product (UshA(Yi)) of the Y. intermedia ushA gene (ushA(Yi)) by its peptide mass fingerprint and by PCR cloning and expression to yield active enzyme. All those activities, except CDP-alcohol hydrolase, have been shown to be the properties of UshA of Escherichia coli (UshA(Ec)). Therefore, UshA(Ec) was expressed from an appropriate plasmid and tested for CDP-alcohol hydrolase activity. UshA(Ec) and UshA(Yi) behaved similarly. Besides being the first study of a UshA enzyme in the genus Yersinia, this work adds CDP-alcohol hydrolase to the spectrum of UshA activities and offers a novel perspective on these proteins, which are viewed here for the first time as highly efficient enzymes with k(cat)/K(m) ratios near the theoretical maximum level of catalytic activities. The results are discussed in the light of the known structures of UshA(Ec) conformers and the respective homology models constructed for UshA(Yi), and also in relation to possible biological functions. Interestingly, every Yersinia species with a sequenced genome contains an intact ushA gene, except Y. pestis, which in all its sequenced biovars contains a ushA gene inactivated by frameshift mutations.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Nucleotidases/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Açúcares de Uridina Difosfato/metabolismo , Yersinia/enzimologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Escherichia coli/química , Escherichia coli/genética , Expressão Gênica , Cinética , Dados de Sequência Molecular , Nucleotidases/química , Nucleotidases/genética , Nucleotidases/isolamento & purificação , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/isolamento & purificação , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Ratos , Especificidade por Substrato , Açúcares de Uridina Difosfato/química , Yersinia/química , Yersinia/genéticaRESUMO
Cyclic ADP-ribose (cADPR) is a messenger for Ca2+ mobilization. Its turnover is believed to occur by glycohydrolysis to ADP-ribose. However, ADP-ribose/CDP-alcohol diphosphatase (ADPRibase-Mn) acts as cADPR phosphohydrolase with much lower efficiency than on its major substrates. Recently, we showed that mutagenesis of human ADPRibase-Mn at Phe37, Leu196 and Cys253 alters its specificity: the best substrate of the mutant F37A + L196F + C253A is cADPR by a short difference, Cys253 mutation being essential for cADPR preference. Its proximity to the 'northern' ribose of cADPR in docking models indicates Cys253 is a steric constraint for cADPR positioning. Aiming to obtain a specific cADPR phosphohydrolase, new mutations were tested at Asp250, Val252, Cys253 and Thr279, all near the 'northern' ribose. First, the mutant F37A + L196F + C253G, with a smaller residue 253 (Ala > Gly), showed increased cADPR specificity. Then, the mutant F37A + L196F + V252A + C253G, with another residue made smaller (Val > Ala), displayed the desired specificity, with cADPR kcat/KM ≈20-200-fold larger than for any other substrate. When tested in nucleotide mixtures, cADPR was exhausted while others remained unaltered. We suggest that the specific cADPR phosphohydrolase, by cell or organism transgenesis, or the designed mutations, by genome editing, provide opportunities to study the effect of cADPR depletion on the many systems where it intervenes.
Assuntos
ADP-Ribosil Ciclase/química , ADP-Ribosil Ciclase/metabolismo , ADP-Ribose Cíclica/química , ADP-Ribose Cíclica/metabolismo , Manganês/química , Manganês/metabolismo , ADP-Ribosilação , ADP-Ribosil Ciclase/genética , Desenho de Fármacos , Ativação Enzimática , Humanos , Ligantes , Modelos Moleculares , Mutação , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Free ADP-ribose is a putative second messenger and also a potentially toxic compound due to its non-enzymic reactivity towards protein side chains. ADP-ribose hydrolysis is catalysed by NDP-sugar/alcohol pyrophosphatases of differing specificity, including a highly specific, low-K(m) ADP-ribose pyrophosphatase. In humans, a submicromolar-K(m) ADP-ribose pyrophosphatase has been purified from placenta, while recombinant NUDT9 has been described as a similarly specific enzyme with a nudix motif, but with a 10(2)-10(3) higher K(m). Here, a comparative study of both proteins is presented showing that they are in fact enzymically indistinguishable; crucially, they both have submicromolar K(m) for ADP-ribose. This study firmly supports the view that the ADP-ribose pyrophosphatase present in human tissues is a product of the NUDT9 gene. In addition, this study reveals previously unknown properties of both enzyme forms. They display the same, differential properties in the presence of Mg(2+) or Mn(2+) as activating cations with respect to substrate specificity, ADP-ribose saturation kinetics, and inhibition by fluoride. Treatment with H(2)O(2) alters the Mg(2+)/Mn(2+) responses and increases the K(m) values for ADP-ribose, changes that are reversed by DTT. The results are discussed in relation to the proposed roles for ADP-ribose in oxidative/nitrosative stress and for ADP-ribose pyrophosphatase as a protective enzyme whose function is to limit the intracellular accumulation of ADP-ribose.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Peróxido de Hidrogênio/farmacologia , Manganês/farmacologia , Placenta/enzimologia , Pirofosfatases/metabolismo , Ditiotreitol/farmacologia , Feminino , Humanos , Cinética , Magnésio/farmacologia , Pirofosfatases/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Endogenous cyclic diadenylate phosphodiesterase activity was accidentally detected in lysates of Escherichia coli BL21. Since this kind of activity is uncommon in Gram-negative bacteria, its identification was undertaken. After partial purification and analysis by denaturing gel electrophoresis, renatured activity correlated with a protein identified by fingerprinting as CpdB (cpdB gene product), which is annotated as 3´-nucleotidase / 2´,3´-cyclic-mononucleotide phosphodiesterase, and it is synthesized as a precursor protein with a signal sequence removable upon export to the periplasm. It has never been studied as a recombinant protein. The coding sequence of mature CpdB was cloned and expressed as a GST fusion protein. The study of the purified recombinant protein, separated from GST, confirmed CpdB annotation. The assay of catalytic efficiencies (kcat/Km) for a large substrate set revealed novel CpdB features, including very high efficiencies for 3´-AMP and 2´,3´-cyclic mononucleotides, and previously unknown activities on cyclic and linear dinucleotides. The catalytic efficiencies of the latter activities, though low in relative terms when compared to the major ones, are far from negligible. Actually, they are perfectly comparable to those of the 'average' enzyme and the known, bona fide cyclic dinucleotide phosphodiesterases. On the other hand, CpdB differs from these enzymes in its extracytoplasmic location and in the absence of EAL, HD and DHH domains. Instead, it contains the domains of the 5´-nucleotidase family pertaining to the metallophosphoesterase superfamily, although CpdB lacks 5´-nucleotidase activity. The possibility that the extracytoplasmic activity of CpdB on cyclic dinucleotides could have physiological meaning is discussed.
Assuntos
2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Nucleotidases/metabolismo , Nucleotídeos Cíclicos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Clonagem Molecular , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Nucleotidases/genética , Diester Fosfórico Hidrolases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type.
Assuntos
Hidrolases Anidrido Ácido/química , Hidrolases Anidrido Ácido/genética , Adenosina Difosfato Ribose/metabolismo , Apirase/química , Apirase/genética , Manganês/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Animais , Apirase/metabolismo , Domínio Catalítico , Humanos , Fígado/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ratos , Homologia Estrutural de ProteínaRESUMO
The ADPRibase-Mn-like protein family, that belongs to the metallo-dependent phosphatase superfamily, has different functional and structural prototypes. The functional one is the Mn(2+)-dependent ADP-ribose/CDP-alcohol diphosphatase from Rattus norvegicus, which is essentially inactive with Mg(2+) and active with low micromolar Mn(2+) in the hydrolysis of the phosphoanhydride linkages of ADP-ribose, CDP-alcohols and cyclic ADP-ribose (cADPR) in order of decreasing efficiency. The structural prototype of the family is a Danio rerio protein with a known crystallographic structure but functionally uncharacterized. To estimate the structure-function correlation with the same protein, the activities of zebrafish ADPRibase-Mn were studied. Differences between zebrafish and rat enzymes are highlighted. The former showed a complex activity dependence on Mn(2+), significant (≈25%) Mg(2+)-dependent activity, but was almost inactive on cADPR (150-fold less efficient than the rat counterpart). The low cADPR hydrolase activity agreed with the zebrafish genome lacking genes coding for proteins with significant homology with cADPR-forming enzymes. Substrate-docking to zebrafish wild-type protein, and characterization of the ADPRibase-Mn H97A mutant pointed to a role of His-97 in catalysis by orientation, and to a bidentate water bridging the dinuclear metal center as the potential nucleophile. Finally, three structural elements that delimit the active site entrance in the zebrafish protein were identified as unique to the ADPRibase-Mn-like family within the metallo-dependent phosphatase superfamily.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Cistina Difosfato/metabolismo , Manganês/metabolismo , Pirofosfatases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra , Animais , Sítios de Ligação , Domínio Catalítico , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Magnésio/farmacologia , Simulação de Acoplamento Molecular , Mutação , Pirofosfatases/química , Pirofosfatases/genética , Ratos , Especificidade por Substrato , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
Cyclic ADP-ribose (cADPR) metabolism in mammals is catalyzed by NAD glycohydrolases (NADases) that, besides forming ADP-ribose, form and hydrolyze the N(1)-glycosidic linkage of cADPR. Thus far, no cADPR phosphohydrolase was known. We tested rat ADP-ribose/CDP-alcohol pyrophosphatase (ADPRibase-Mn) and found that cADPR is an ADPRibase-Mn ligand and substrate. ADPRibase-Mn activity on cADPR was 65-fold less efficient than on ADP-ribose, the best substrate. This is similar to the ADP-ribose/cADPR formation ratio by NADases. The product of cADPR phosphohydrolysis by ADPRibase-Mn was N(1)-(5-phosphoribosyl)-AMP, suggesting a novel route for cADPR turnover.
Assuntos
Adenosina Difosfato Ribose/química , ADP-Ribose Cíclica/química , Manganês/metabolismo , Pirofosfatases/química , Adenosina Difosfato Ribose/metabolismo , Animais , ADP-Ribose Cíclica/metabolismo , Hidrólise , Modelos Moleculares , Pirofosfatases/metabolismo , Ratos , Especificidade por SubstratoRESUMO
We have investigated the biological occurrence of riboflavin 4',5'-cyclic phosphate (cyclic FMN or cFMN), the flavin product known to be formed in vitro from FAD by the rat liver enzyme FAD-AMP lyase (cyclizing) or FMN cyclase (EC 4.6.1.15). The expected difficulties were the low concentration of the compound, the tendency of the more abundant FAD to decompose chemically to cFMN, and the acid lability of cFMN itself. A protocol was devised to prepare deproteinized rat liver extracts, avoiding conditions prone to the chemical formation of cFMN and making exposure to conditions of cFMN degradation as short as possible. In these extracts, cFMN was assayed by HPLC with fluorimetric detection. The identity of liver cFMN was confirmed by its HPLC separation from other known flavins, its coelution with authentic cFMN, and its susceptibility to acid degradation, yielding a mixture of 5'-FMN and 4'-FMN. The amount of total cFMN recovered in the liver extracts was 22+/-11 pmol/g fresh tissue. Careful control experiments were performed to rule out the possibility that this could be a chemical product of FAD degradation during extract preparation. These controls indicated that, on average, 97% of the measured extract concentration of cFMN, i.e., about 21+/-10 pmol/g, was already present in the liver at the beginning of the process and was extracted from the tissue. This figure is likely to be an underestimation of the hepatic content, as indicated by control experiments.