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  • The PCR product was expressed under T promoter

    2020-07-30

    The PCR product was expressed under T7 promoter in E. coli, yielding transformants with 180-fold higher PPase activity than the host strain, clearly indicating that the open reading frame encodes a PPase. Like authentic B. subtilis PPase [22], the recombinant enzyme purified to homogeneity was activated by preincubation with Mn2+ and Co2+ (Fig. 1A), but not with Mg2+, Ca2+, Sr2+, Cd2+, Cu2+, Fe2+ or Ni2+. Full activation was obtained in 2 min of incubation. The activation was strongly dependent on metal ion concentration (Fig. 1A) and pH (Fig. 1B). Maximal activation was observed at pH 8.5 with 1.5 mM Mn2+ (Fig. 1). At pH>9, manganese hydroxide precipitated, limiting our ability to study the activation at high pH values. The specific activity of the enzyme at pH 7.3 was 323 and 9900 U/mg before and after the activation by 1.5 mM Mn2+, corresponding to the kcat values of 180 and 5500 s−1, respectively. Interestingly, the former kcat value is about the same as those for E. coli and yeast PPases 11, 12, whereas the latter is greater by an order of magnitude. According to its amino protein kinase sequence, B. subtilis PPase is unique among known soluble PPases. There are 31 soluble PPase sequences currently available in the GenBank: prokaryotic PPases have 164–233 amino acid residues per subunit, whereas eukaryotic PPases have 211–310 residues per subunit. In this respect, B. subtilis PPase, with 309 amino acid residues per subunit, resembles eukaryotic PPases. Furthermore, B. subtilis PPase shows little sequence similarity to other soluble PPases, typical examples of which are shown in Fig. 2. Sequence identity between PPase of B. subtilis and E. coli is as low as 17%, whereas internal identity of the 21 available prokaryotic PPase sequences varies from 31% (Haemophilus influenzae vs. E. coli) to 61% (Legionella pneumophila vs. E. coli). Fan rom evolutionary point of view, it is interesting to note that Bacillus stearothermophilus, a close relative of B. subtilis, has a PPase which is very similar to other soluble PPases [28], but completely different from B. subtilis PPase (Fig. 2): identities of B. stearothermophilus PPase vs. E. coli PPase and B. subtilis PPase are 44 and 15%, respectively. An even more striking difference is observed when comparing active site residues, which are evolutionarily very well conserved in other soluble PPases 18, 19. In 31 known PPase sequences, 13 functionally important active site residues are conserved in all sequences, but only two of them (D70 and D97 in E. coli PPase numbering) are conserved in B. subtilis PPase (Fig. 2). In addition to soluble PPases, plants and certain bacteria have a membrane-bound PPase, which works as a reversible proton pump. Membrane-bound PPases differ in many respects from soluble PPases – they protein kinase are much larger (660–770 amino acid residues per monomer) and do not have any sequence similarity to soluble PPases 29, 30, 31. The B. subtilis PPase described here is clearly a soluble PPase, and it does not have any sequence similarity to membrane-bound PPases.