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  • The presence of H PPases in parasitic protists


    The presence of H+-PPases in parasitic protists raises the question of the physiological role of these proteins. So far, they had been extensively characterized mainly in higher plants, and some prokaryotes [1], [2], [3], [4], [5], [6], [7], [16], [20]. A common feature to all these organisms is that they are adapted to different sorts of stress in their natural habitats: shortage of nutrients (e.g., phosphate in plants), dim-light photosynthesis in R547 conditions (photosynthetic proteobacteria) or extreme environments (archaea and hyperthermophilic bacteria). This could also be the case of protozoan parasites, because they are subjected to stressing conditions by the host defenses. In these tough circumstances all these organisms may have some difficulty in obtaining the energy needed for surviving, therefore they have developed (or retained) the ability to generate an electrochemical proton gradient, a very useful and versatile form of biological energy, from an abundant by-product of anabolism, such as PPi. It is relevant to note in this respect that in L. major promastigotes the intracellular level of PPi increased concurrently with a decrease of long-chain polyphosphates under metabolic stressing conditions, i.e., in the absence of exogenous carbon source and/or anaerobiosis [35]. The fact that this strategy has not been followed by many other organisms, including fungi and animals, points towards H+-PPases as potential targets for therapeutic agents (vaccines and drugs) that could be used against diseases that claim a high number of human lives worldwide.
    Introduction Inorganic pyrophosphatase (EC catalyzes specifically the hydrolysis of pyrophosphate to orthophosphate. This reaction provides a thermodynamic pull for many biosynthetic reactions 1, 2, 3and is essential for life 4, 5, 6. PPases require bivalent metal ions for catalysis, with Mg2+ conferring the highest activity. The best-studied PPases are those from Escherichia coli and Saccharomyces cerevisiae, which have been extensively characterized by X-ray crystallography 7, 8, 9, 10and site-directed mutagenesis in combination with kinetic and thermodynamic measurements 11, 12, 13, 14, 15, 16, 17. All known soluble PPases are homologous proteins, whose active site structure is evolutionarily very well conserved 18, 19. In the 1960s, Tono and Kornberg [20]carried out initial characterization of soluble R547 PPase from Bacillus subtilis, and recently the enzyme was characterized in more detail 21, 22. In contrast to other soluble PPases, B. subtilis PPase is activated by preincubation with Mn2+ ions, which presumably affect the equilibrium between the active trimer and the less active dimer [22]. In the present study, the gene encoding B. subtilis PPase was cloned and expressed in E. coli, the enzyme was compared in terms of amino acid sequence with other soluble PPases and shown to be a first member of a new, Bs family of soluble PPases.
    Materials and methods Restriction endonucleases, T4 DNA polymerase, Taq polymerase and a DNA ligation kit were purchased from Takara Shuzo, [α-32P]dCTP (3000 Ci/mmol) and a DNA sequencing kit were from Amersham, a Gene Clean II kit was from Bio 101, plasmid vector pET-3a was from Novagen, marker proteins for SDS-PAGE were from Bio-Rad, phenyl-Sepharose CL-4B was from Pharmacia and DEAE-HPLC column was from Tosoh. B. subtilis strain AC327 was used throughout this study. E. coli JM109 [23]and E. coli BL21(DE3) (Novagen) were used as hosts in the cloning and expression, respectively. The strains were grown in LB broth or on 2×YT plates [23]. Ampicillin (50 μg/ml) was added when required. B. subtilis PPase was expressed in E. coli under the inducible phage T7 promoter by making use of the pET system (Novagen). The open reading frame encoding B. subtilis PPase was amplified by polymerase chain reaction (PCR) using a 5′-sense oligonucleotide primer containing a restriction site for NdeI (5′-GCGGAAAAGATACTTATTTTCG-3′, the restriction site is underlined) and a 3′-reverse complement primer with a BamHI site (5′-AATTATTCAGCCATTGCGTCT-3′). The PCR product was digested with NdeI and BamHI, ligated into the vector pET-3a (Novagen), cut with the same restriction enzymes and transformed into E. coli JM109 and E. coli BL21(DE3) for DNA sequencing [24]and expression, respectively. The expression was induced for 3–6 h by 0.5 mM IPTG.