Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi

Abstract

A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K m values being 11.8 to 56.3 M. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K i and K i values of 0.455 to 11.2 M. Metal ion chelators inhibited activity, and the addition of Zn 2؉ or Co 2؉ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH 2 -terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.Recently, nucleosides and a variety of chemically synthesized nucleoside analogs have attracted a great deal of interest, as they have antibiotic, antiviral, and antitumoral effects (9). In light of this trend, we conducted studies on the microbial metabolism of nucleosides (5). In this study, we found that a bacterium, Ochrobactrum anthropi, shows a high level of activity in the N-riboside cleavage of purine nucleosides. This reaction is important in the decomposition of purine nucleosides in foodstuffs which cause hyperuricemia, an increasingly common disease in adults (3).The enzymatic N-riboside cleavage of nucleosides is a common reaction in various organisms (1,20). This reaction seems to participate in a salvage or assimilation pathway for nucleosides. Two kinds of enzymes, nucleoside phosphorylases (EC 2.4.2.-) and nucleosidases (nucleoside hydrolase; EC 3.2.2.-), are known to catalyze this reaction. Nucleoside phosphorylases catalyze the phosphorolytic cleavage of nucleosides and show ribosyl transferase activity (7). These enzymes play roles mainly in the salvage pathway. Nucleoside phosphorylases have been well studied. In addition, they have been purified from various sources and used as catalysts for the synthesis of nucleoside analogs through base exchange reactions (7, 21). In contrast, nucleosidases catalyze the irreversible hydrolysis of nucleosides and participate mainly in the assimilation pathway.There have been few studies on microbial nucleosidases acting on purine and pyrimidine nucleosides and no reports of homogenously purified bacterial purine and pyrimidine nucleosidases (8,18,19).In this study, we report the...

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“…Nucleoside phosphorylase activities resulting in N-riboside cleavage of purine nucleosides have been identified in various organisms (29). We observed that feeding xanthosine to our yeast cultures results in the production of xanthine, a product synthesized through riboside cleavage of xanthosine.…”

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“…We first investigated the quaternary structure of recombinant TTHA0556 because the purine nucleosidases so far examined are reported to form dimers, tetramers, and pentamers. [6][7][8][9][10][11] An assay mixture containing the purified MBP-fused recombinant (Fig. 2) with a calculated molecular mass of 66.3 kDa (MBP; 42.5 kDa, TTHA0556; 23.8 kDa) was subjected to gel filtration.…”

“…[6][7][8][9]12) Various bases and compounds, which were used to examine substrate specificity, as described above, were tested at a final concentration of 0.5 mM under standard assay conditions. Of these, only hypoxanthine showed an inhibitory effect, and this was weak (the specific activity fell to approximately 80% of that without the additive).…”

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“…7) As part of this trend, the microbial metabolism of nucleosides has been intensively investigated. [8][9][10][11][12] The first reaction in nucleoside metabolism is N-riboside cleavage. Two kinds of enzymes, nucleosidase (nucleoside hydrolase; EC 3.2.2.-) and nucleoside phosphorylase (EC 2.4.2.-), are known to catalyze this reaction (Fig.…”