Purification and Characterization of Second Thermostable Purine Nucleoside Phosphorylase inBacillus stearothermophilusJTS 859

Abstract

The isoelectric point was 5.3. The Michaelis constants for inosine, guanosine, and adenosine were 0.22, 0.34, and 0.075 mM, respectively. The optimal temperature of the reaction was 70°C. The enzyme was stable at 70°C. Although other reported purine nucleoside phosphorylases were SH-enzymes, ,PUNPII was not a SH-enzyme because the enzyme reaction was not inhibited by PCMB and iodoacetic acid, the optimal pH of the enzyme reaction was from 7.0 to 11.0, and the enzyme did not contain cysteine. PUNPII and PUNPI were different in several points. Not PUNPI but PUNPII could catalyze the phosphorolysis of adenosine. Specific activity of PUNPI and II for inosine were 405 and 50.6,umol/min/mg protein at 60°C, respectively. PUNPI was stable at 80°C. PUNPII was stable at 70°C, but was denatured at 80°C.

Molecular cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of a purine nucleoside phosphorylase fromBacillus subtilisstrain 168

“…Hexameric PNPs are prevalent in prokaryotes and exhibit a broader range of substrates. In some microorganisms, such as Escherichia coli and Bacillus spp., both a trimeric and a hexameric form of PNP are found (Seeger et al, 1995;Senesi et al, 1976;Hori et al, 1989).…”

Crystal structure ofEscherichia colipurine nucleoside phosphorylase complexed with acyclovir

“…The other members of family I, mammalian PNPs, have a trimeric structure and are specific for 6-oxopurine ribonucleosides only. In some microorganisms, such as E. coli and Bacillus subtilis, both trimeric and hexameric forms of PNP are found (Seeger et al, 1995;Senesi et al, 1976;Hori et al, 1989). An alignment of the amino-acid sequences of several representatives of both types of PNP is shown in Fig.…”

Cloning of Purine Nucleoside Phosphorylase II Gene fromBacillus stearothermophilusTH 6–2 and Characterization of Its Gene Product

“…The characterization Pu-NPase II showed that the enzyme closely resembled the second type of nucleoside phosphorylase isolated from B. stearothermophilus JTS 859. 18 ) Thus we concluded that the cloned punB gene encoded the Pu-NPase II.…”