Purification and characterization of second thermostable purine nucleoside phosphorylase in Bacillus stearothermophilus JTS 859.

Abstract

Thermostable purine nucleoside phosphorylases, PUNPI and PUNPII, have been purified from Bacillus stearothermophilus JTS 859. The characterization of PUNPIwas reported previously. [Hori et al, Agric. Biol Chem. 53, 2205] PUNPII had a molecular weight of 113,000, consisting of 4 identical subunits (Mw28,000). 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, PUNPIIwas not a SH-enzymebecause the enzyme reaction was not inhibited by PCMBand iodoacetic acid, the optimal pH of the enzyme reaction was from 7.0 to ll.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|imol/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.Purine nucleoside phosphorylase (PUNP) (EC. 2.4.2. 1) catalyzes the reversible phosphorylation of ribonucleoside and deoxyribonucleoside derivatives of hypoxanthine, guanine, and xanthine as follows: purine nucleoside + phosphate <± purine base+pentose-1 -phosphate Since the substrate specificity of PUNPis not strict, the enzymehas been used to prepare purine nucleoside analogues, such as hypoxanthine arabinoside, virazole, and 6-mercaptopurine nucleoside.1~4)Wehave studied the practical application of ribosyl transfer reaction by thermostable PUNP and pyrimidine nucleoside phosphorylase (PYNP) (EC 2.4.2.2) at high temperatures.5) Wereported the purification and the characterization of a thermostable PUNP, PUNPI from Bacillus stearothermophilus JTS 859, and also the existence of another PUNP, PUNPII, in our previous paper.6) This paper 3219 describes the purification and the characterization of PUNPII. These two enzymes were different in their substrate specificity, specific activity, and thermal stability. Not PUNPI but PUNPII could catalyze the phosphorolysis of adenosine. The specific activities of PUNPI and II for insoine were 405 and 50.6 /xmol/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. Materials and MethodsMicroorganism. Bacillus stearothermophilus JTS 859, which produced thermostable purine nucleoside phosphorylases, was used as the enzyme source.Chemicals. Inosine was purchased from Yamasa Chemicals (Tokyo).Peptone A and yeast extract were purchased from Kyokuto Co. (Tokyo).Culture conditions. Medium,containing (g/1 of water) Peptone A 20, yeast extract 10, glucose 3, inosine 1, and

Structural analyses reveal two distinct families of nucleoside phosphorylases

“…Mammalian species generally possess only the trimeric form of PNP. Bacterial species have largely been shown to possess only the hexameric form, although both a trimeric and a hexameric form have been found in E. coli [47], Bacillus subtilis [48] and Bacillus stearothermophilus [49].…”

The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology

“…Sequence analysis among PNPs suggests that there are two major categories of this enzyme. Mammalian PNPs, such as those from human erythrocytes and bovine spleen, are trimeric and have a monomeric molecular weight of 31 kDa [1][2][3][4], whereas prokaryotic PNPs are hexameric with a monomeric molecular weight of 26 kDa [5][6][7][8][9][10][11]. In general, PNPs with a trimeric structure accept only guanosine and inosine as substrates, whereas the hexameric PNPs accept adenosine as well, although in at least one instance a hexameric PNP is specific for adenosine and deoxyadenosine [9].…”

“…In addition, Escherichia coli PNP is more tolerant of nucleosides that contain modified ribose moieties, and compared to mammalian PNP its activity toward purine arabinosides is ten times higher [12]. Interestingly, both trimeric and hexameric PNPs have been observed in Bacillus subtilis [8], E. coli [10] and Bacillus stearothermophilus [11].…”

Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36

“…This would also apply to the BHPNP1 protein. However, gel filtration analysis of the G. stearothermophilus PNP1 gave an apparent molecular weight of 68,000, potentially indicating a dimeric protein (Hori et al 1989b ).…”

“…In addition, thermophiles have been shown to harbour enzymes that exhibit a much greater thermostability. For example, the thermophile Geobacillus stearothermophilus (previously Bacillus stearothermophilus ) has two purine nucleoside phosphorylases which have been characterised (Hori et al 1989a , b ; Saunders et al 1969 ) and applied in the synthesis of 5-methyluridine (Hori et al 1989c , 1991 ). However, although these enzymes are thermostable, they have low levels of expression in the wild type.…”