Rosa María Pinto

Specific ADP-ribose pyrophosphatase from Artemia cysts and rat liver: effects of nitroprusside, fluoride and ionic strength

One specific ADP-ribose pyrophosphatase (ADPRibase) has been identified in Artemia cysts, following a protocol that in rat liver allows the identification of three ADPRibases. Artemia ADPRibase resulted similar, but not identical, to rat liver ADPRibase-I with respect to known and novel properties disclosed in this work. In the presence of Mg2+, Artemia ADPRibase was highly specific for ADP-ribose and showed a low, 0.7 microM Km. Preincubation with the nitric oxide donor nitroprusside and dithiothreitol, elicited dose- and time-dependent, severalfold increase of Km and decrease of Vmax. At saturating ADP-ribose concentrations, fluoride was a strong inhibitor (IC50 approximately equal to 10-20 microM), whereas bringing ionic strength to 0.3-1.3 mol/l doubled the activity measured at lower or higher strengths. The novel fluoride and ionic strength effects were studied also with rat liver ADPRibase-I. Differences between the Artemia enzyme and ADPRibase-I concerned molecular weight (31,000 versus 38,500, respectively), Mn2+ ability to substitute for Mg2+ as the activating cation (better for the rat enzyme), and Vmax decrease by nitroprusside (not seen with the rat enzyme). The results are discussed in relation with the role of specific ADPRibases as protective factors limiting free ADP-ribose accumulation and protein glycation, and as targets for cytotoxic agents.

Location of dinucleoside triphosphatase in the matrix space of rat liver mitochondria

The submitochondrial location of dinucleoside triphosphatase (EC 3.6.1.29). previously shown to be in part associated with mitochondria, has been studied in rat liver. The precipitability and latency of activity in organelle suspensions, and the profile of solubilization by digitonin, were like those of the matrix space marker glutamate dehydrogenase, and differed from those other submitochondrial fractions. This, and the synthesis of diadenosine polyphosphates by mitochondrial aminoacyl‐tRNA synthetases, suggest the occurrence of a pathway for the intramitochondrial turnover of diadenosine 5′,5‴‐P 1,P 3‐triphosphate (Ap2A).

IMP dehydrogenase from Artemia embryos: Molecular forms, purification and properties

Abstract 1. 1. IMP dehydrogenase (EC 1.2.1.14) has been purified near homogeneity from Artemia embryos. 2. 2. The K m values for IMP and NAD + were 15 and 200 μM, respectively. 3. 3. GMP, XMP, GTP, guanosine 5′-tetraphosphate and diguanosine tetraphosphate (Gp 4 G) were competitive inhibitors of the reaction towards IMP with K i values of 140, 180, 175, 120 and 87 μM, respectively. 4. 4. The enzyme from the 27,000 g supernatant can occur in a number of oligomeric forms (450, 375, 260 or 220 kDa) depending on the ionic strength of the medium. 5. 5. Upon precipitation with ammonium sulphate (0.3–0.4 saturation) the enzyme aggregates forming complexes of more than 1000 kDa.

The Characterization of Escherichia coli CpdB as a Recombinant Protein Reveals that, besides Having the Expected 3´-Nucleotidase and 2´,3´-Cyclic Mononucleotide Phosphodiesterase Activities, It Is Also Active as Cyclic Dinucleotide Phosphodiesterase.

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.

Bifunctional homodimeric triokinase/FMN cyclase: contribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movements.

Background: Triokinase, which phosphorylates dihydroxyacetone and fructose-derived glyceraldehyde, remains molecularly unidentified. Results: Human DAK gene encodes homodimeric triokinase/FMN cyclase formed by two-domain subunits. Although kinase activity requires intact homodimers, cyclase requires only a truncated, single domain subunit. Conclusion: Triokinase/FMN cyclase identity and bifunctionality are established. Significance: This study molecularly dissects a bifunctional enzyme of unusual specificity and finishes the molecular identification of fructose pathway enzymes. 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 Thr112 (hydrogen bonding of ATP adenine to K in the closed active center), His221 (covalent anchoring of dihydroxyacetone to K), Asp401 and Asp403 (metal coordination to L), and Asp556 (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His221 point mutant acted specifically as a cyclase without kinase activity.

CGDEase, a Pseudomonas fluorescens protein of the PLC/APase superfamily with CDP-ethanolamine and (dihexanoyl)glycerophosphoethanolamine hydrolase activity induced by osmoprotectants under phosphate-deficient conditions

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.

Molecular bases of catalysis and ADP-ribose preference of human Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase and conversion by mutagenesis to a preferential cyclic ADP-ribose phosphohydrolase.

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 K m and unchanged k cat of F37A-ADPRibase-Mn, while the K m 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.

Adenosine deaminase isozymes in Artemia

Three isozymes of adenosine deaminase, termed II, III and IV, have been detected in Artemia embryos. Their pI values, determined by chromatofocusing, were 4.9, 5.0 and 5.2, respectively. Upon development to larvae, a different isozyme (I) is induced, with a pI value of 4.2 as determined by isoelectric focusing.

Presence of two isozymes of adenylosuccinate synthetase in Artemia salina embryos. Purification and properties

Abstract 1. 1. Two isozymes of adenylosuccinate synthetase, termed I and II have been separated by chromatography on DEAE-cellulose (DE-52), in Artemia embryos. 2. 2. The following data refer to form I and II: apparent molecular weights determined by sucrose gradient centrifugation and chromatography on Sephadex G-100: (I) 96,700, 90,000 and (II) 97,800, 83,000; K m values for their three substrates IMP, GTP and aspartate: (I) 26, 29, 600 and (II) 20, 25, 700 μM; fructose 1,6-biphosphate was a competitive inhibitor towards IMP with a K i value of 22 μM for both isozymes. 3. 3. The relationship between the Artemia enzyme and that from rat liver and other sources, is also discussed in this paper.