José Carlos Cameselle

A specific, low Km ADP-ribose pyrophosphatase from rat liver

Two rat liver ADP‐ribose pyrophosphatases (ADPRibases) were partially purified. ADPRibase‐I hydrolyzed ADP‐ribose (K m=0.5 μM) giving AMP as a product, required Mg2+ or, less efficiently, Mn2+ (Ca2+ was not active), its activity changed little between pH 7 and 9, and was specific for ADP‐ribose as it did not hydrolyze ADP‐glucose, NAD+, NADH or diadenosine 5′,5″‐P 1,P n ‐n‐phosphates (Ap2A, Ap3A). ADPRibase‐II showed similar properties, except that the K m for ADP‐ribose was 50 μM and may be non‐specific, as the same preparation hydrolyzed ADP‐glucose, NADH and Ap2A. ADPRibase‐I fulfils the requirements of a specific turnover pathway consistent with a cellular role for free ADP‐ribose.

Glycine-enhanced inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase-I by EDTA: a full account of the reported inhibition by commercial preparations of acidic fibroblast growth factor (FGF-1).

The earlier reported inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase I (EC 3.1.6.9/EC 3.1.4.1; NPP/PDE) by culture‐grade acidic fibroblast growth factor (FGF‐1) correlates with a low‐M r contaminant. 1H‐NMR analyses revealed EDTA in the total‐volume fractions of a gel‐filtration experiment, where all the inhibitory activity of the FGF‐1 preparation was recovered. NPP/PDE inhibition by EDTA (and by unfractionated FGF‐1 or the EDTA‐containing fractions) was time‐dependent, blocked by the substrate p‐nitrophenyl‐dTMP, and strongly enhanced by glycine. The use of glycine buffers in earlier work was critical to the apparent inhibition by FGF‐1. The results point to a conformational change favored by glycine that may be relevant to the biological role of NPP/PDE.

Hydrolysis of the phosphoanhydride linkage of cyclic ADP-ribose by the Mn2+-dependent ADP-ribose/CDP-alcohol pyrophosphatase

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.

ADP‐ribose pyrophosphatase‐I partially purified from livers of rats overdosed with acetaminophen reveals enzyme inhibition in vivo reverted in vitro by dithiothreitol

Free ADP‐ribose reacts nonenzymatically with proteins and can lead to intracellular damage. The low‐Km ADP‐ribose pyrophosphatase‐I (ADPRibase‐I) is well suited to control free ADP‐ribose and nonenzymatic ADP‐ribosylation. In vitro, the acetaminophen metabolite N‐acetyl‐p‐benzoquinoneimine (NAPQI) decreases ADPRibase‐I Vmax and increases Km, effects not reverted by dithiothreitol (DTT) and attributed to enzyme arylation. The present study was conducted to test whether acetaminophen overdose affected ADPRibase‐I in vivo. Rats pretreated with 3‐methylcholanthrene and L‐buthionine‐[S,R]‐sulfoximine to potentiate acetaminophen toxicity received an intraperitoneal dose of either acetaminophen (800 mg/kg; n = 5) or vehicle (n = 3). ADPRibase‐I partially purified from acetaminophen‐overdosed rats showed a decreased Vmax (0.32 ± 0.09 versus 0.60 ± 0.03 mU/mg of liver protein; p < 0.01) not reverted by DTT and an increased Km for ADP‐ribose (1.39 ± 0.31 versus 0.67 ± 0.05 μM; p < 0.01) that, contrary to the in vitro NAPQI effect, was reverted by DTT. Incubation of partially purified ADPRibase‐I from normal rat liver with oxidized glutathione elicited a time‐ and dose‐dependent, DTT‐reverted increase of Km, without change of Vmax. The results indicate that the activity of ADPRibase‐I can be regulated by thiol exchange and that the increase of Km elicited by acetaminophen overdosage was related to the oxidative stress caused by the drug. It remains to be seen whether an increase of free ADP‐ribose concomitant to ADPRibase‐I inhibition could contribute to the hepatotoxicity of acetaminophen.

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).

Rat liver ADP-ribose pyrophosphatase-I as an in vitro target of the acetaminophen metabolite N-acetyl-p-benzoquinoneimine

N-acetyl-p-benzoquinoneimine (NAPQI) is the metabolite responsible for acetaminophen hepatotoxicity. ADP-ribose pyrophosphatase-I (ADPRibase-I; EC 3.6.1.13) hydrolyzes protein-glycating ADP-ribose. The results show NAPQI-dependent alterations of ADPRibase-I leading to strong inhibition: a fast Km increase produced by low concentrations, and a time-dependent Vmax decrease by higher NAPQI concentrations. Both effects were prevented by thiols, but not reverted by them, nor by gel filtration of NAPQI-treated enzyme. Liver ADPRibase-I can be a target of NAPQI-dependent arylation. The inhibition or inactivation of the enzyme would contribute to increasing the free ADP-ribose concentration and nonenzymatic ADP-ribosylation, which is coherent with results linking free ADP-ribose-producing pathways to acetaminophen toxicity.

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.

Characterization of Danio rerio Mn2+-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase, the Structural Prototype of the ADPRibase-Mn-Like Protein Family

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 Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase from Rattus norvegicus, which is essentially inactive with Mg2+ and active with low micromolar Mn2+ 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 Mn2+, significant (≈25%) Mg2+-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.

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.