Jared L. Cartwright

THE SACCHAROMYCES CEREVISIAE YOR163W GENE ENCODES A DIADENOSINE 5', 5-P1, P6-HEXAPHOSPHATE (AP6A) HYDROLASE MEMBER OF THE MUTT MOTIF (NUDIX HYDROLASE) FAMILY

The YOR163w open reading frame on chromosome XV of the Saccharomyces cerevisiae genome encodes a member of the MutT motif (nudix hydrolase) family of enzymes ofM r 21,443. By cloning and expressing this gene in Escherichia coli and S. cerevisiae, we have shown the product to be a (di)adenosine polyphosphate hydrolase with a previously undescribed substrate specificity. Diadenosine 5′,5‴-P 1,P 6-hexaphosphate is the preferred substrate, and hydrolysis in H2 18O shows that ADP and adenosine 5′-tetraphosphate are produced by attack at Pβ and AMP and adenosine 5′-pentaphosphate are produced by attack at Pα with a K m of 56 μmand k cat of 0.4 s−1. Diadenosine 5′,5‴-P 1,P 5-pentaphosphate, adenosine 5′-pentaphosphate, and adenosine 5′-tetraphosphate are also substrates, but not diadenosine 5′,5‴-P 1,P 4-tetraphosphate or other dinucleotides, mononucleotides, nucleotide sugars, or nucleotide alcohols. The enzyme, which was shown to be expressed in log phase yeast cells by immunoblotting, displays optimal activity at pH 6.9, 50 °C, and 4–10 mm Mg2+ (or 200 μm Mn2+). It has an absolute requirement for a reducing agent, such as dithiothreitol (1 mm), and is inhibited by Ca2+ with an IC50 of 3.3 mm and F− (noncompetitively) with aK i of 80 μm. Its function may be to eliminate potentially toxic dinucleoside polyphosphates during sporulation.

The Saccharomyces cerevisiae PCD1 Gene Encodes a Peroxisomal Nudix Hydrolase Active toward Coenzyme A and Its Derivatives

The PCD1 nudix hydrolase gene ofSaccharomyces cerevisiae has been cloned and the Pcd1p protein characterized as a diphosphatase (pyrophosphatase) with specificity for coenzyme A and CoA derivatives. Oxidized CoA disulfide is preferred over CoA as a substrate with K m andk cat values of 24 μm and 5.0 s− 1, respectively, compared with values for CoA of 280 μm and 4.6 s− 1respectively. The products of CoA hydrolysis were 3′-phosphoadenosine 5′-monophosphate and 4′-phosphopantetheine. F− ions inhibited the activity with an IC50 of 22 μm. The sequence of Pcd1p contains a potential PTS2 peroxisomal targeting signal. When fused to the N terminus of yeast-enhanced green fluorescent protein, Pcd1p was shown to locate to peroxisomes by confocal microscopy. It was also shown to co-localize with peroxisomal thiolase by immunofluorescence microscopy. N-terminal sequence analysis of the expressed protein revealed the loss of 7 or 8 amino acids, suggesting processing of the proposed PTS2 signal after import. The function of Pcd1p may be to remove potentially toxic oxidized CoA disulfide from peroxisomes in order to maintain the capacity for β-oxidation of fatty acids.

The g5R (D250) Gene of African Swine Fever Virus Encodes a Nudix Hydrolase That Preferentially Degrades Diphosphoinositol Polyphosphates

ABSTRACT The African swine fever virus (ASFV) g5R gene encodes a protein containing a Nudix hydrolase motif which in terms of sequence appears most closely related to the mammalian diadenosine tetraphosphate (Ap4A) hydrolases. However, purified recombinant g5R protein (g5Rp) showed a much wider range of nucleotide substrate specificity compared to eukaryotic Ap4A hydrolases, having highest activity with GTP, followed by adenosine 5′-pentaphosphate (p5A) and dGTP. Diadenosine and diguanosine nucleotides were substrates, but the enzyme showed no activity with cap analogues such as 7mGp3A. In common with eukaryotic diadenosine hexaphosphate (Ap6A) hydrolases, which prefer higher-order polyphosphates as substrates, g5Rp also hydrolyzes the diphosphoinositol polyphosphates PP-InsP5 and [PP]2-InsP4. A comparison of the kinetics of substrate utilization showed that the k cat/K m ratio for PP-InsP5 is 60-fold higher than that for GTP, which allows classification of g5R as a novel diphosphoinositol polyphosphate phosphohydrolase (DIPP). Unlike mammalian DIPP, g5Rp appeared to preferentially remove the 5-β-phosphate from both PP-InsP5 and [PP]2-InsP4. ASFV infection led to a reduction in the levels of PP-InsP5, ATP and GTP by ca. 50% at late times postinfection. The measured intracellular concentrations of these compounds were comparable to the respective K m values of g5Rp, suggesting that one or all of these may be substrates for g5Rp during ASFV infection. Transfection of ASFV-infected Vero cells with a plasmid encoding epitope-tagged g5Rp suggested localization of this protein in the rough endoplasmic reticulum. These results suggest a possible role for g5Rp in regulating a stage of viral morphogenesis involving diphosphoinositol polyphosphate-mediated membrane trafficking.

GTP:GTP guanylyltransferase: Trapping procedures for detecting and characterizing chemical nature of enzyme-nucleotide phosphoramidate reaction intermediate

This chapter focuses on trapping procedures for detecting and characterizing the chemical nature of enzyme-nucleotide phosphoramidate reaction intermediate. The chemical nature of the enzyme-nucleotide phosphoramidate intermediate employed by the unique GTP:GTP guanylyltransferase from yolk platelets of Artemia franciscana cysts to synthesize diguanosine tetraphosphate (Gp4G) has been investigated. The chapter describes the formation and trapping of the enzyme-guanylate reaction intermediate along with experiments to establish the identity of the bound nucleotide and to determine that it represents a genuine reaction intermediate. The chemical stability of the intermediate yields information about the nature of the covalent linkage and may even suggest the identity of the amino acid involved. The intermediate in the ligases and capping enzymes involves an enzyme lysyl residue, while the others employ a histidyl residue. In the absence of sequence and structural information, as is the case with the Artemia Gp4G synthetase, characterization of the reaction intermediate can help in the tentative allocation of an enzyme to a particular protein family.