Activation of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus by removal of magnesium inhibition and acceleration of product release .

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.

Part-time alpha-secretases: the functional biology of ADAM 9, 10 and 17.

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.

Structural and mutational analysis of substrate complexation by anthranilate phosphoribosyltransferase from Sulfolobus solfataricus.

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.