1887

Abstract

This work reports the evolutionary relationships, amplified expression, functional characterization and purification of the putative allantoin transport protein, PucI, from . Sequence alignments and phylogenetic analysis confirmed close evolutionary relationships between PucI and membrane proteins of the nucleobase-cation-symport-1 family of secondary active transporters. These include the sodium-coupled hydantoin transport protein, Mhp1, from , and related proteins from bacteria, fungi and plants. Membrane topology predictions for PucI were consistent with 12 putative transmembrane-spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. The gene was cloned into the IPTG-inducible plasmid pTTQ18 upstream from an in-frame hexahistidine tag and conditions determined for optimal amplified expression of the PucI(His) protein in to a level of about 5 % in inner membranes. Initial rates of inducible PucI-mediated uptake of C-allantoin into energized whole cells conformed to Michaelis–Menten kinetics with an apparent affinity ( app) of 24 ± 3 μM, therefore confirming that PucI is a medium-affinity transporter of allantoin. Dependence of allantoin transport on sodium was not apparent. Competitive uptake experiments showed that PucI recognizes some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides. PucI(His) was solubilized from inner membranes using n-dodecyl-β--maltoside and purified. The isolated protein contained a substantial proportion of α-helix secondary structure, consistent with the predictions, and a 3D model was therefore constructed on a template of the Mhp1 structure, which aided localization of the potential ligand binding site in PucI.

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2016-05-01
2024-03-28
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References

  1. Adelman J. L., Dale A. L., Zwier M. C., Bhatt D., Chong L. T., Zuckerman D. M., Grabe M. 2011; Simulations of the alternating access mechanism of the sodium symporter Mhp1. Biophys J 101:2399–2407 [View Article][PubMed]
    [Google Scholar]
  2. Arora R., Papaioannou V. E. 2012; The murine allantois: a model system for the study of blood vessel formation. Blood 120:2562–2572 [View Article][PubMed]
    [Google Scholar]
  3. Beier L., Nygaard P., Jarmer H., Saxild H. H. 2002; Transcription analysis of the Bacillus subtilis PucR regulon and identification of a cis-acting sequence required for PucR-regulated expression of genes involved in purine catabolism. J Bacteriol 184:3232–3241 [View Article][PubMed]
    [Google Scholar]
  4. Bernsel A., Viklund H., Hennerdal A., Elofsson A. 2009; TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Res 37:W465–W468 [View Article][PubMed]
    [Google Scholar]
  5. Bettaney K. E., Sukumar P., Hussain R., Siligardi G., Henderson P. J., Patching S. G. 2013; A systematic approach to the amplified expression, functional characterization and purification of inositol transporters from Bacillus subtilis . Mol Membr Biol 30:3–14 [View Article][PubMed]
    [Google Scholar]
  6. Biasini M., Bienert S., Waterhouse A., Arnold K., Studer G., Schmidt T., Kiefer F., Gallo Cassarino T., Bertoni M., other authors. 2014; SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42:W252–W258 [View Article][PubMed]
    [Google Scholar]
  7. Christiansen L. C., Schou S., Nygaard P., Saxild H. H. 1997; Xanthine metabolism in Bacillus subtilis: characterization of the xpt-pbuX operon and evidence for purine- and nitrogen-controlled expression of genes involved in xanthine salvage and catabolism. J Bacteriol 179:2540–2550[PubMed]
    [Google Scholar]
  8. Cruz-Ramos H., Glaser P., Wray L. V. Jr., Fisher S. H. 1997; The Bacillus subtilis ureABC operon. J Bacteriol 179:3371–3373[PubMed]
    [Google Scholar]
  9. Danielsen S., Boyd D., Neuhard J. 1995; Membrane topology analysis of the Escherichia coli cytosine permease. Microbiology 141:2905–2913 [View Article][PubMed]
    [Google Scholar]
  10. de Koning H., Diallinas G. 2000; Nucleobase transporters. Mol Membr Biol 17:75–94 [View Article][PubMed]
    [Google Scholar]
  11. Emsley P., Cowtan K. 2004; Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132 [View Article][PubMed]
    [Google Scholar]
  12. Findlay H. E., Rutherford N. G., Henderson P. J., Booth P. J. 2010; Unfolding free energy of a two-domain transmembrane sugar transport protein. Proc Natl Acad Sci U S A 107:18451–18456 [View Article][PubMed]
    [Google Scholar]
  13. Fisher S. H. 1993; Utilization of amino acids and other nitrogen-containing compounds. In Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics pp 221–228 Edited by Sonenshein A. L., Hoch J. A., Losick R. Washington, DC: American Society for Microbiology; [View Article]
    [Google Scholar]
  14. Fisher S. H. 1999; Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!. Mol Microbiol 32:223–232 [View Article][PubMed]
    [Google Scholar]
  15. Goelzer A., Bekkal Brikci F., Martin-Verstraete I., Noirot P., Bessières P., Aymerich S., Fromion V. 2008; Reconstruction and analysis of the genetic and metabolic regulatory networks of the central metabolism of Bacillus subtilis . BMC Syst Biol 2:20 [View Article][PubMed]
    [Google Scholar]
  16. Goudela S., Karatza P., Koukaki M., Frillingos S., Diallinas G. 2005; Comparative substrate recognition by bacterial and fungal purine transporters of the NAT/NCS2 family. Mol Membr Biol 22:263–275 [View Article][PubMed]
    [Google Scholar]
  17. Guex N., Peitsch M. C. 1997; SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723 [View Article][PubMed]
    [Google Scholar]
  18. Hamari Z., Amillis S., Drevet C., Apostolaki A., Vágvölgyi C., Diallinas G., Scazzocchio C. 2009; Convergent evolution and orphan genes in the Fur4p-like family and characterization of a general nucleoside transporter in Aspergillus nidulans . Mol Microbiol 73:43–57 [View Article][PubMed]
    [Google Scholar]
  19. Henderson P. J., Giddens R. A., Jones-Mortimer M. C. 1977; Transport of galactose, glucose and their molecular analogues by Escherichia coli K12. Biochem J 162:309–320 [CrossRef]
    [Google Scholar]
  20. Inman K. E., Downs K. M. 2007; The murine allantois: emerging paradigms in development of the mammalian umbilical cord and its relation to the fetus. Genesis 45:237–258 [View Article][PubMed]
    [Google Scholar]
  21. Jackson S. M., Patching S. G., Ivanova E., Simmons K. J., Weyand S., Shimamura T., Brueckner F., Suzuki S., Iwata S., other authors. 2013; Mhp1, the Na+-hydantoin membrane transport protein. In Encyclopedia of Biophysics pp 1514–1521 Edited by Roberts G. C. K. Berlin, Heidelberg: Springer; [View Article]
    [Google Scholar]
  22. Johnson R. J., Sautin Y. Y., Oliver W. J., Roncal C., Mu W., Sanchez-Lozada L. G., Rodriguez-Iturbe B., Nakagawa T., Benner S. A. 2009; Lessons from comparative physiology: could uric acid represent a physiologic alarm signal gone awry in western society?. J Comp Physiol B 179:67–76 [View Article][PubMed]
    [Google Scholar]
  23. Kazmier K., Sharma S., Islam S. M., Roux B., Mchaourab H. S. 2014; Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1. Proc Natl Acad Sci U S A 111:14752–14757 [View Article][PubMed]
    [Google Scholar]
  24. Krogh A., Larsson B., von Heijne G., Sonnhammer E. L. 2001; Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580 [View Article][PubMed]
    [Google Scholar]
  25. Krypotou E., Evangelidis T., Bobonis J., Pittis A. A., Gabaldón T., Scazzocchio C., Mikros E., Diallinas G. 2015; Origin, diversification and substrate specificity in the family of NCS1/FUR transporters. Mol Microbiol 96:927–950 [View Article][PubMed]
    [Google Scholar]
  26. Laskowski R. A., Swindells M. B. 2011; LigPlot+: multiple ligand–protein interaction diagrams for drug discovery. J Chem Inf Model 51:2778–2786 [View Article][PubMed]
    [Google Scholar]
  27. Lovell S. C., Davis I. W., Arendall W.B., III, de Bakker P. I., Word J. M., Prisant M. G., Richardson J. S., Richardson D. C. 2003; Structure validation by Cα geometry: φ, ψ and Cβ deviation. Proteins 50:437–450 [View Article][PubMed]
    [Google Scholar]
  28. Ma P., Yuille H. M., Blessie V., Göhring N., Iglói Z., Nishiguchi K., Nakayama J., Henderson P. J. F., Phillips-Jones M. K. 2008; Expression, purification and activities of the entire family of intact membrane sensor kinases from Enterococcus faecalis . Mol Membr Biol 25:449–473 [View Article][PubMed]
    [Google Scholar]
  29. Ma P., Varela F., Magoch M., Silva A. R., Rosário A. L., Brito J., Oliveira T. F., Nogly P., Pessanha M., other authors. 2013; An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli . PLoS One 8:e76913 [View Article][PubMed]
    [Google Scholar]
  30. Mourad G. S., Tippmann-Crosby J., Hunt K. A., Gicheru Y., Bade K., Mansfield T. A., Schultes N. P. 2012; Genetic and molecular characterization reveals a unique nucleobase cation symporter 1 in Arabidopsis . FEBS Lett 586:1370–1378 [View Article][PubMed]
    [Google Scholar]
  31. Nygaard P., Saxild H. H. 2005; The purine efflux pump PbuE in Bacillus subtilis modulates expression of the PurR and G-box (XptR) regulons by adjusting the purine base pool size. J Bacteriol 187:791–794 [View Article][PubMed]
    [Google Scholar]
  32. Nygaard P., Duckert P., Saxild H. H. 1996; Role of adenine deaminase in purine salvage and nitrogen metabolism and characterization of the ade gene in Bacillus subtilis . J Bacteriol 178:846–853[PubMed]
    [Google Scholar]
  33. Nygaard P., Bested S. M., Andersen K. A. K., Saxild H. H. 2000; Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source. Microbiology 146:3061–3069 [View Article][PubMed]
    [Google Scholar]
  34. Pantazopoulou A., Diallinas G. 2007; Fungal nucleobase transporters. FEMS Microbiol Rev 31:657–675 [View Article][PubMed]
    [Google Scholar]
  35. Patching S. G. 2009; Synthesis of highly pure 14C-labelled DL-allantoin and 13C NMR analysis of labelling integrity. J Labelled Comp Radiopharm 52:401–404 [View Article]
    [Google Scholar]
  36. Pizzichini M., Pandolfi M. L., Arezzini L., Terzuoli L., Fe L., Bontemps F., Van den Berghe G., Marinello E. 1996; Labelling of uric acid and allantoin in different purine organs and urine of the rat. Life Sci 59:893–899 [View Article][PubMed]
    [Google Scholar]
  37. Rapp M., Schein J., Hunt K. A., Nalam V., Mourad G. S., Schultes N. P. 2016; The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility. Protoplasma 253:611–623[PubMed] [CrossRef]
    [Google Scholar]
  38. Rath A., Glibowicka M., Nadeau V. G., Chen G., Deber C. M. 2009; Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proc Natl Acad Sci U S A 106:1760–1765 [View Article][PubMed]
    [Google Scholar]
  39. Saidijam M., Patching S. G. 2015; Amino acid composition analysis of secondary transport proteins from Escherichia coli with relation to functional classification, ligand specificity and structure. J Biomol Struct Dyn 33:2205–2220 [View Article][PubMed]
    [Google Scholar]
  40. Saidijam M., Psakis G., Clough J. L., Meuller J., Suzuki S., Hoyle C. J., Palmer S. L., Morrison S. M., Pos M. K., other authors. 2003; Collection and characterisation of bacterial membrane proteins. FEBS Lett 555:170–175 [View Article][PubMed]
    [Google Scholar]
  41. Saxild H. H., Brunstedt K., Nielsen K. I., Jarmer H., Nygaard P. 2001; Definition of the Bacillus subtilis PurR operator using genetic and bioinformatic tools and expansion of the PurR regulon with glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO . J Bacteriol 183:6175–6183 [View Article][PubMed]
    [Google Scholar]
  42. Schein J. R., Hunt K. A., Minton J. A., Schultes N. P., Mourad G. S. 2013; The nucleobase cation symporter 1 of Chlamydomonas reinhardtii and that of the evolutionarily distant Arabidopsis thaliana display parallel function and establish a plant-specific solute transport profile. Plant Physiol Biochem 70:52–60 [View Article][PubMed]
    [Google Scholar]
  43. Schultz A. C., Nygaard P., Saxild H. H. 2001; Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator. J Bacteriol 183:3293–3302 [View Article][PubMed]
    [Google Scholar]
  44. Shi Y. 2013; Common folds and transport mechanisms of secondary active transporters. Annu Rev Biophys 42:51–72 [View Article][PubMed]
    [Google Scholar]
  45. Shimamura T., Weyand S., Beckstein O., Rutherford N. G., Hadden J. M., Sharples D., Sansom M. S., Iwata S., Henderson P. J., Cameron A. D. 2010; Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1. Science 328:470–473 [View Article][PubMed]
    [Google Scholar]
  46. Sievers F., Wilm A., Dineen D., Gibson T. A., Karplus K., Li W., Lopez R., McWilliam H., Remmert M., other authors. 2011; Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539 [CrossRef]
    [Google Scholar]
  47. Simmons K. J., Jackson S. M., Brueckner F., Patching S. G., Beckstein O., Ivanova E., Geng T., Weyand S., Drew D., other authors. 2014; Molecular mechanism of ligand recognition by membrane transport protein, Mhp1. EMBO J 33:1831–1844 [View Article][PubMed]
    [Google Scholar]
  48. Stark M. J. R. 1987; Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in Escherichia coli . Gene 51:255–267 [View Article][PubMed]
    [Google Scholar]
  49. Suzuki S., Henderson P. J. 2006; The hydantoin transport protein from Microbacterium liquefaciens . J Bacteriol 188:3329–3336 [View Article][PubMed]
    [Google Scholar]
  50. Szakonyi G., Leng D., Ma P., Bettaney K. E., Saidijam M., Ward A., Zibaei S., Gardiner A. T., Cogdell R. J., other authors. 2007; A genomic strategy for cloning, expressing and purifying efflux proteins of the major facilitator superfamily. J Antimicrob Chemother 59:1265–1270 [View Article][PubMed]
    [Google Scholar]
  51. von Heijne G. 1992; Membrane protein structure prediction: hydrophobicity analysis and the positive-inside rule. J Mol Biol 225:487–494 [View Article][PubMed]
    [Google Scholar]
  52. Ward A., O'Reilly J., Rutherford N. G., Ferguson S. M., Hoyle C. K., Palmer S. L., Clough J. L., Venter H., Xie H., other authors. 1999; Expression of prokaryotic membrane transport proteins in Escherichia coli . Biochem Soc Trans 27:893–899 [View Article][PubMed]
    [Google Scholar]
  53. Ward A., Sanderson N. M., O'Reilly J., Rutherford N. G., Poolman B., Henderson P. J. F. 2000; The amplified expression, identification, purification, assay and properties of hexahistidine-tagged bacterial membrane transport proteins. In Membrane transport – a Practical Approach pp 141–166 Edited by Baldwin S. A. Oxford: Blackwell;
    [Google Scholar]
  54. Weyand S., Shimamura T., Yajima S., Suzuki S., Mirza O., Krusong K., Carpenter E. P., Rutherford N. G., Hadden J. M., other authors. 2008; Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter. Science 322:709–713 [View Article][PubMed]
    [Google Scholar]
  55. Weyand S., Ma P., Saidijam M., Baldwin J., Beckstein O., Jackson S., Suzuki S., Patching S. G., Shimamura T., other authors. 2010; The nucleobase-cation-symport-1 family of membrane transport proteins. In Handbook of Metalloproteins 11 Edited by Messerschmidt A. Chichester: Wiley; [View Article]
    [Google Scholar]
  56. Weyand S., Shimamura T., Beckstein O., Sansom M. S., Iwata S., Henderson P. J., Cameron A. D. 2011; The alternating access mechanism of transport as observed in the sodium-hydantoin transporter Mhp1. J Synchrotron Radiat 18:20–23 [View Article][PubMed]
    [Google Scholar]
  57. Witz S., Panwar P., Schober M., Deppe J., Pasha F. A., Lemieux M. J., Möhlmann T. 2014; Structure-function relationship of a plant NCS1 member – homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis . PLoS One 9:e91343 [View Article][PubMed]
    [Google Scholar]
  58. Wray L.V., Jr, Ferson A. E., Rohrer K., Fisher S. H. 1996; TnrA, a transcription factor required for global nitrogen regulation in Bacillus subtilis . Proc Natl Acad Sci U S A 93:8841–8845 [View Article][PubMed]
    [Google Scholar]
  59. Xi H., Schneider B. L., Reitzer L. 2000; Purine catabolism in Escherichia coli and function of xanthine dehydrogenase in purine salvage. J Bacteriol 182:5332–5341 [View Article][PubMed]
    [Google Scholar]
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