The metabolic synthesis and degradation of essential nucleotide compounds are primarily carried out by phosphoribosyltransferases (PRT) and nucleoside phosphorylases (NP), respectively. Despite the resemblance of their reactions, five classes of PRTs and NPs exist, where anthranilate PRT (AnPRT) constitutes the only evolutionary link between synthesis and degradation processes. We have characterized the active site of dimeric AnPRT from Sulfolobus solfataricus by elucidating crystal structures of the wild-type enzyme complexed to its two natural substrates anthranilate and 5-phosphoribosyl-1-pyrophosphate/Mg 2؉ . These bind into two different domains within each protomer and are brought together during catalysis by rotational domain motions as shown by small angle x-ray scattering data. Steady-state kinetics of mutated AnPRT variants address the role of active site residues in binding and catalysis. Results allow the comparative analysis of PRT and pyrimidine NP families and expose related structural motifs involved in nucleotide/ nucleoside recognition by these enzyme families.Nucleotide compounds are central to the production of genetic material, the amino acids histidine and tryptophan, and cofactors such as NAD. Their metabolism is largely based on the reversible transfer of a phosphoribosyl group to aromatic bases. Although forward and reverse ribosylation processes share a close resemblance (Fig. 1), they are carried out by different enzymes and in the context of distinct metabolic pathways. Synthesis reactions are performed by phosphoribosyltransferases (PRT). 3 These use PRPP as universal phosphoribosyl donor. These reactions, which are dependent on metal ions, involve the displacement of the 1-pyrophosphate group of PRPP and formation of a N-1Ј-glycosidic bond to a nitrogenated base specific for each PRT. Conversely, the release of aromatic bases from nucleotides involves first the production of a nucleoside intermediate by nucleotidases or phosphatases, followed by the cleavage of the glycosidic bond by either nucleoside phosphorylases (NP) or nucleoside hydrolases. NP enzymes are key to nucleotide salvage both in prokaryotes and in eukaryotes. They catalyze the reversible phosphorolysis of the glycosidic bond of nucleosides to yield free bases and ribose-1-phosphate.Despite the chemical resemblance of the compounds intervening in synthesis and degradation reactions by PRTs and NPs ( Fig. 1), respectively, the enzymes involved in these catalyses lack structural similarity. Three diverse PRT classes that act on aromatic bases are known to date, as follows: (i) those with a canonical fold classified as type PRT-I, which includes most of the PRTs for which an atomic structure is available (1); (ii) type PRT-II comprising quinolate PRT (2-3) and nicotinate PRT (4); and (iii) anthranilate PRT (AnPRT) (5-7) (Fig. 1A). NPs are also classified in two groups with distinct folds as follows: (i) NP-I, which act primarily on purine nucleosides but also accept the pyrimidine uridine, and (ii) NP-II, which degrade t...

Disintegrin metalloproteases of the ADAM family form a large (at present > 40 members in mammals) family of multidomain membrane proteins that in their ectodomain combine a cystein-rich, disintegrin and a zinc metalloprotease domain. Via their metalloprotease domain, ADAMs are often implicated in ectodomain shedding, either to release e.g. growth factors or to initiate further intracellular signalling via regulated intramembrane proteolysis. Mainly based upon overexpression studies in vehicle cells, three of them, ADAMs 9, 10 and 17, have been proposed to act as alpha-secretases for amyloid precursor protein (APP). It is striking thereby that this role has since then remained somewhat ill-defined, as APP processing in ADAM9 deficient neurons is unaltered, and also ADAM10 deficient murine embryonic fibroblasts exhibit at best a highly variable reduction in alpha-secretase activity. However, during the past years, numerous other substrates have been linked to all three sheddases, the cleavage of which in some cases appears to be strikingly more important for the organism than APP processing. Most notably, the perinatally lethal phenotype of ADAM17 knockout mice is dominated by a loss of growth factor shedding, while the even earlier fatal effects of ADAM10 deficiency exhibit key features of disabled Notch signalling and possibly also cadherin processing defects. In this review, we will summarize the published data on the "non-APP" functions of all three ADAMs, the further evaluation of which may be crucial when attempting to treat Alzheimer s Disease by increasing their expression and/or activity. As the knockouts of ADAM10 and ADAM17 are only informative for their roles in (early) development, while a number of recently assigned new substrates play crucial roles in the normal and/or diseased adult organism as well, work on conditional knockout models will be crucial to fully characterize both the full functional portfolio of (candidate) alpha-secretases as well as their clinical relevance, which may go way beyond Alzheimer s Disease.

Anthranilate phosphoribosyltransferase from the hyperthermophilic archaeon Sulfolobus solfataricus (ssAnPRT) is encoded by the sstrpD gene and catalyzes the reaction of anthranilate (AA) with a complex of Mg(2+) and 5'-phosphoribosyl-alpha1-pyrophosphate (Mg.PRPP) to N-(5'-phosphoribosyl)-anthranilate (PRA) and pyrophosphate (PP(i)) within tryptophan biosynthesis. The ssAnPRT enzyme is highly thermostable (half-life at 85 degrees C = 35 min) but only marginally active at ambient temperatures (turnover number at 37 degrees C = 0.33 s(-1)). To understand the reason for the poor catalytic proficiency of ssAnPRT, we have isolated from an sstrpD library the activated ssAnPRT-D83G + F149S double mutant by metabolic complementation of an auxotrophic Escherichia coli strain. Whereas the activity of purified wild-type ssAnPRT is strongly reduced in the presence of high concentrations of Mg(2+) ions, this inhibition is no longer observed in the double mutant and the ssAnPRT-D83G single mutant. The comparison of the crystal structures of activated and wild-type ssAnPRT shows that the D83G mutation alters the binding mode of the substrate Mg.PRPP. Analysis of PRPP and Mg(2+)-dependent enzymatic activity indicates that this leads to a decreased affinity for a second Mg(2+) ion and thus reduces the concentration of enzymes with the inhibitory Mg(2).PRPP complex bound to the active site. Moreover, the turnover number of the double mutant ssAnPRT-D83G + F149S is elevated 40-fold compared to the wild-type enzyme, which can be attributed to an accelerated release of the product PRA. This effect appears to be mainly caused by an increased conformational flexibility induced by the F149S mutation, a hypothesis which is supported by the reduced thermal stability of the ssAnPRT-F149S single mutant.