Purification and properties of inosine-guanosine phosphorylase from Escherichia coli K-12

Abstract

A xanthosine-inducible enzyme, inosine-guanosine phosphorylase, has been partially purified from a strain of Escherichia coli K-12 lacking the deo-encoded purine nucleoside phosphorylase. Inosine-guanosine phosphorylase had a particle weight of 180 kilodaltons and was rapidly inactivated by p-chloromercuriphenylsulfonic acid (p-CMB). The enzyme was not protected from inactivation by inosine (Ino), 2'-deoxyinosine (dlno), hypoxanthine (Hyp), Pi, or c-D-ribose-1-phosphate (Rib-1-P). Incubating the inactive enzyme with dithiothreitol restored the catalytic activity. Reaction with p-CMB did not affect the particle weight. Inosine-guanosine phosphorylase was more sensitive to thermal inactivation than purine nucleoside phosphorylase. The half-life determined at 45°C between pH 5 and 8 was 5 to 9 min. Phosphate (20 mM) stabilized the enzyme to thermal inactivation, while Ino (1 mM), dIno (1 mM), xanthosine (Xao) (1 mM), Rib-i-P (2 mM), or Hyp (0.05 mM) had no effect. However, Hyp at 1 mM did stabilize the enzyme. In addition, the combination of P1 (20 mM) and Hyp (0.05 mM) stabilized this enzyme to a greater extent than did Pi alone. Apparent activation energies of 11.5 kcal/mol and 7.9 kcal/mol were determined in the phosphorolytic and synthetic direction, respectively. The pH dependence of Ino cleavage or synthesis did not vary between 6 and 8. The substrate specificity, listed in decreasing order of efficiency (V/Ki), was: 2'-deoxyguanosine, dIno, guanosine, Xao, Ino, 5'-dIno, and 2',3'-dideoxyinosine. Inosine-guanosine phosphorylase differed from the deo operon-encoded purine nucleoside phosphorylase in that neither adenosine, 2'-deoxyadenosine, nor hypoxanthine arabinoside were substrates or potent inhibitors. Moreover, the E. coli inosine-guanosine phosphorylase was antigenically distinct from the purine nucleoside phosphorylase since it did not react with any of 14 monoclonal antisera or a polyvalent antiserum raised against deo-encoded purine nucleoside phosphorylase.Nucleoside phosphorylases catalyze the reversible phosphorolysis of either purine or pyrimidine nucleosides, yielding pentose-1-phosphate and the nucleobase. Three nucleoside phosphorylases expressed by Escherichia coli have been well characterized (10,12,20,24). The udp gene product, uridine phosphorylase (EC 2.4.2.3), and the deoA gene product, thymidine phosphorylase (EC 2.4.2.4), catalyze the phosphorolysis and synthesis of pyrimidine ribonucleosides and 2'-deoxyribonucleosides, respectively. The third enzyme, purine nucleoside phosphorylase (E.C. 2.4.2.1), encoded by the deoD gene of the deo operon, cleaves and synthesizes both purine ribonucleosides and 2'-deoxyribonucleosides.The nucleobase specificity of purine nucleoside phosphorylase is dependent on the source from which it is isolated. Hyp, Gua, Xan, and their respective nucleosides are efficient substrates for most mammalian purine nucleoside phosphorylases, whereas Ade and Ade nucleosides are extremely inefficient substrates (14,28,32). The nucleobase specificity of purine nucleoside...

Genes encoding ribonucleoside hydrolase 1 and 2 from Corynebacterium ammoniagenes

“…In Escherichia coli, exogenous ribonucleosides are predominantly metabolized by nucleoside phosphorylases encoded by deoD, udp and xapA, while NHs encoded by rihA, rihB and rihC have been reported and play a minor role (Koszalka et al, 1988;Petersen & Moller, 2001). In the case of the genus Bacillus, the metabolic pathways mediated by only nucleoside phosphorylases have been reported and characterized (Hamamoto et al, 1996;Rocchietti et al, 2004), whereas in the yeast Saccharomyces cerevisiae, purine ribonucleosides, inosine and guanosine, and pyrimidine ribonucleosides, cytidine and uridine, were salvaged by purine nucleoside phosphorylase (encoded by pnp1) and uridine ribohydrolase (encoded by urh1), respectively (Desgranges et al, 2001;Kurtz et al, 2002;Mitterbauer et al, 2002).…”

Thermus thermophilus Nucleoside Phosphorylases Active in the Synthesis of Nucleoside Analogues

“…They are generally classified attending to their substrate specificity as purine NPs (PNPs; EC 2.4.2.1) or pyrimidine NPs (PyNPs; EC 2.4.2.2). Other classifications regarding their molecular mass and structure have also been made (4), although several NPs have been reported to deviate from these proposed classes (6)(7)(8)14). It is not possible to establish a clear relationship between a given sequence or structure and its nucleoside specificity.…”

Identification and characterization of genes (xapA, xapB, and xapR) involved in xanthosine catabolism in Escherichia coli

“…Two of these genes, xapA and xapR, have previously been characterized genetically (8), and xanthosine phosphorylase (XapA) has been purified and characterized (12,24). The finding of the xapB gene, however, was quite unexpected.…”

“…In contrast to this, the xanthosine phosphorylase activity can be measured only when cells are grown in the presence of xanthosine, and the xanthosine phosphorylase activity is not influenced by DeoR or CytR. Xanthosine phosphorylase has been purified and partly characterized (3,12,24).…”