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Purine nucleoside phosphorylase (PNP ; EC 2.4.2.1) from many bacteria, including E. coli and some pathogenic organisms, functions as a homohexamer, composed of three linked dimers.1, 2 It is a promising candidate for a tumour directed gene therapy, a target to design antiparasitic drugs and a very useful tool for enzymatic synthesis of nucleosides. We investigated the reasons underlying the necessity of the hexameric form, since it appears that the dimer has all the necessary structural features to conduct catalysis.3-6 X- ray diffraction studies revealed that in the apo form of the E. coli enzyme all six subunits have the same structure with entry into the active site open.3, 4 According to the accepted molecular mechanism of catalysis, closing of the active site has to occur that brings catalytic Arg217 close to the Asp204, enabling protonation of purine ring of the nucleoside.6 X-ray structure shows4, in line with solution studies7, that this conformational change occurs already in the binary complex of PNP with phosphate, but only in two neighboring monomers belonging to different dimers. The third dimer keeps its original open conformation of both active sites4. The same is observed in some ternary complexes of E. coli PNP with substrate analogues.5 But in just one such structure the alternate arrangement of monomers with open and closed active sites appears.6 In the H/D exchange mass spectrometry the averaged deuterium uptake reaches only 32% of all exchangeable hydrogen atoms, pointing to a high rigidity of the hexamer. The deuterium incorporation mapped onto the PNP tertiary structure shows only a few flexible regions at the molecule surface and within the dimer interface, while the interface between dimers and the middle of the hexamer are very rigid.8 Molecular modelling was employed to probe mutations in the region of the dimer-dimer interface that would result in stable dimers from the hexameric structure. In this way, by mutation of 3, 4 and 6 residues, the hexamer was successfully transformed into dimers, confirmed by analytical ultracentrifugation.9 However, CD spectra reveal slight changes in the secondary structure elements, and the catalytic activity is negligible. The modelling indicate that in solution the isolated dimer cannot maintain the appropriate three- dimensional structure, including the geometry of the active site and the position of the catalytically important amino acids.9 We conclude that the dimer-dimer interactions in the hexameric molecule of E. coli PNP are necessary to provide stabilization of the proper for catalysis three-dimensional arrangement of the dimeric structure. It is still to be answered are all three dimers stabilized in this way, or only two, with the third dimer keeping the open-open conformation even in the ternary complex with substrates, not conducting catalysis and serving as a scaffold for the remaining two. 1. A Bzowska, E Kulikowska, D Shugar, Pharmacol Ther 88, 349- 425 (2000) 2. Y Zhang, WB Parker, EJ Sorscher, et al. Curr Topics in Med Chem 5, 1259-1274 (2005) 3. C Mao, WJ Cook, M Zhou, GW Koszalka, et al. Structure. 5, 1373-1383 (1997) 4. G Mikleušević, Z Štefanić, M Narczyk, et al. Biochimie 93, 1610-1622 (2011) 5. EM Bennett, C Li PW Allan, BW Parker, et al. J Biol Chem 278, 47110-47118 (2003) 6. G Koellner, A Bzowska, B Wielgus-Kutrowska, et al., J Mol. Biol 315, 351-371 (2002) 7. B Kierdaszuk, A Modrak- Wójcik, D Shugar, Biophys Chem 63, 107-118 (1997) 8. S Kazazić, B Bertoša, et al. Amer Soc Mass Spec DOI: 10.1007/s13361-015-1239-2 (2015) 9. B Bertoša, G Mikleušević, B Wielgus- Kutrowska, et al. FEBS J. 281, 1860-1871 (2014) |
