Free amino acids accelerate the time-dependent inactivation of rat liver nucleotide pyrophosphatase/phosphodiesterase Enpp3 elicited by EDTA.

Nucleotide-pyrophosphatases/phosphodiesterases (NPP/PDE) are membrane or secreted Zn-metallohydrolases of nucleoside-5´-monophosphate derivatives. They hydrolyze, for instance, ATP and 4-nitrophenyl-dTMP, and belong to the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family that contains seven members (ENPP1-ENPP7). Earlier we had shown that an NPP/PDE activity solubilized and partially purified from rat liver membranes is inactivated by EDTA in a time-dependent fashion, an effect enhanced by glycine and blocked by the 4-nitrophenyl-dTMP.

Alternative Splicing of the Last Intron Yields Transcripts Differentially Expressed in Human Tissues That Code In Vitro for a Protein Devoid of Triokinase and FMN Cyclase Activity.

The 18-exon human gene codes for dual-activity triokinase and FMN cyclase (TKFC) in an ORF, spanning from exon 2 to exon 18. In addition to TKFC-coding transcripts (classified as type by their intron-17 splice), databases contain evidence for alternative transcripts, but none of them has been expressed, studied, and reported in the literature. A novel full-ORF transcript was cloned from brain cDNA and sequenced (accession no.

Homozygous variant in abolishing triokinase activities is associated with isolated immunodeficiency.

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).

Genomic Distribution of -like Genes in : Comparison to -like Genes.

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.

Genomic Distribution of Pro-Virulent -like Genes in Eubacteria and Comparison of the Enzyme Specificity of CpdB-like Proteins from , and .

The gene is pro-virulent in avian pathogenic and in , 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 and genes of and , respectively. CdnP and SntA effects are due to extrabacterial hydrolysis of cyclic-di-AMP, and to complement action interference.

Enzyme Characterization of Pro-virulent SntA, a Cell Wall-Anchored Protein of , With Phosphodiesterase Activity on cyclic-di-AMP at a Level Suited to Limit the Innate Immune System.

and 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 TKFC Ala185Thr variant, reported as 'null' for fructose metabolism, is fully active as triokinase.

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.

Substrate Specificity of Chimeric Enzymes Formed by Interchange of the Catalytic and Specificity Domains of the 5-Nucleotidase UshA and the 3-Nucleotidase CpdB.

The 5'-nucleotidase UshA and the 3'-nucleotidase CpdB from 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.

Molecular Dissection of CpdB: Roles of the N Domain in Catalysis and Phosphate Inhibition, and of the C Domain in Substrate Specificity and Adenosine Inhibition.

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-β response in infected hosts. The gene of Gram-negative and its homologs of gram-positive bacteria are virulence factors.

Autosomal recessive hypotrichosis with loose anagen hairs associated with TKFC mutations.

Background: Loose anagen hair is a rare form of impaired hair anchorage in which anagen hairs that lack inner and outer root sheaths can be gently and painlessly plucked from the scalp. This condition usually occurs in children and is often self-limiting. A genetic basis for the disorder has been suggested but not proven.

Closure of the Human TKFC Active Site: Comparison of the Apoenzyme and the Complexes Formed with Either Triokinase or FMN Cyclase Substrates.

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.

TERTp mutation is associated with a shorter progression free survival in patients with aggressive histology subtypes of follicular-cell derived thyroid carcinoma.

Purpose: Evaluate the impact of TERTp mutation on the outcomes after initial treatment of 45 patients with thyroid carcinomas derived from follicular cells (TCDFC) with aggressive histology, in which the role of this mutation is not yet well defined.

Methods: Analysis of the presence of TERTp (-124C > T and -146C > T), BRAF (V600E), and NRAS (Q 61R) mutations by Sanger sequencing and analysis of their correlation with the patient's outcomes.

Results: Forty-five patients with aggressive histopathologic variants were included in the study.

Specific cyclic ADP-ribose phosphohydrolase obtained by mutagenic engineering of Mn-dependent ADP-ribose/CDP-alcohol diphosphatase.

Cyclic ADP-ribose (cADPR) is a messenger for Ca 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.

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.

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.

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

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.

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 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.

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.

A bifunctional molecule as an artificial flavin mononucleotide cyclase and a chemosensor for selective fluorescent detection of flavins.

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.

Hydrolysis of the phosphoanhydride linkage of cyclic ADP-ribose by the Mn(2+)-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.

CDP-alcohol hydrolase, a very efficient activity of the 5'-nucleotidase/UDP-sugar hydrolase encoded by the ushA gene of Yersinia intermedia and Escherichia coli.

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.

Mn2+-dependent ADP-ribose/CDP-alcohol pyrophosphatase: a novel metallophosphoesterase family preferentially expressed in rodent immune cells.

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.

The specific, submicromolar-Km ADP-ribose pyrophosphatase purified from human placenta is enzymically indistinguishable from recombinant NUDT9 protein, including a selectivity for Mn2+ as activating cation and increase in Km for ADP-ribose, both elicited by H2O2.

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).

Identification of human and rat FAD-AMP lyase (cyclic FMN forming) as ATP-dependent dihydroxyacetone kinases.

Rat liver FAD-AMP lyase or FMN cyclase is the only known enzymatic source of the unusual flavin nucleotide riboflavin 4',5'-cyclic phosphate. To determine its molecular identity, a peptide-mass fingerprint of the purified rat enzyme was obtained. It pointed to highly related, mammalian hypothetical proteins putatively classified as dihydroxyacetone (Dha) kinases due to weaker homologies to biochemically proven Dha kinases of plants, yeasts, and bacteria.

Fluorimetric HPLC detection of endogenous riboflavin 4',5'-cyclic phosphate in rat liver at nanomolar concentrations.

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.