1887

Abstract

The genes for the proton-translocating nicotinamide nucleotide transhydrogenase from have been cloned using a probe constructed with the polymerase chain reaction, genomic DNA as target and oligonucleotide primers corresponding to amino acid sequence obtained from the purified soluble subunit. There is a cluster of three genes, designated and , whose translation products indicate polypeptides of 384, 139 and 464 amino acids, respectively. This contrasts with the situation in the enzymes from (two polypeptides) and bovine mitochondria (one polypeptide) but there is close similarity between the sequences. PntAA is the soluble subunit of the enzyme from , equivalent to the relatively hydrophilic domain I that forms the N-terminal part of the α polypeptide of transhydrogenase and which probably contains the NAD(H)-binding site. PntAB corresponds to the strongly hydrophobic domain IIa at the C-terminus of the α polypeptide of the transhydrogenase. PntB corresponds to the β polypeptide, which comprises the strongly hydrophobic domain IIb and the relatively hydrophilic domain III, thought to contain the NADP(H)-binding site. The peptide bond between PntAA-Lys237 and -Glu238 of both the denatured and the native soluble subunit is very sensitive to proteolysis by trypsin and the neighbouring peptide bond Lys227-Thr228 to cleavage by the endoproteinase Lys-C. Related sites have been reported to be sensitive to trypsin in the and bovine mitochondrial enzymes. The two tryptic fragments from the native soluble subunit are unable to reconstitute transhydrogenase activity to membranes depleted of the soluble subunit but they can block reconstitution by intact soluble subunit. It is suggested that this protease-sensitive region separates two subdomains and that, after trypsinolysis, at least one retains structural integrity and can dock with domains II and/or III.

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1994-07-01
2024-11-13
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References

  1. Adams M.D., Dubnick M., Kerlavage A.R., Moreno R., Kelley J.M., Utterback T.R., Nagle J.W., Fields C., Venter J.C. Sequence identification of 2375 human brain genes. Nature 1992; 355:632–634
    [Google Scholar]
  2. Ahmad S., Glavas N.A., Bragg P.D. A mutation at Gly314 of the (1 subunit of the Escherichia coli pyridine nucleotide transhydrogenase abolishes activity and affects the NADP(H)-induced conformational change. Eur J biochem 1992; 207:733–739
    [Google Scholar]
  3. Bimboim H.C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 1979; 7:1513–1523
    [Google Scholar]
  4. Clarke D.M., Loo T.W., Gillam S., Bragg P.D. Nucleotide sequence of the put A and pntb genes encoding the pyridine nucleotide transhydrogenase of Escherichia coli. Eur J biochem 1986; 158:647–653
    [Google Scholar]
  5. Clayton R.K. Towards the isolation of a photochemical reaction centre in Rhodopseudomonas capsulata. Biochim Biophys Acta 1963; 73:312–323
    [Google Scholar]
  6. Cunningham I.J., Williams R., Palmer T., Thomas C.M., Jackson J.B. The relation between the soluble factor associated with H+-transhydrogenase of Rhodospirillum rubrum and the enzyme from mitochondria and Escherichia coli. Biochim Biophys Acta 1992a; 1100:332–338
    [Google Scholar]
  7. Cunningham I.J., Baker J.A., Jackson J.B. Reaction between the soluble and membrane-associated proteins of the transhvdrogenase of Rhodospirillum rubrum. Biochim Biophys Acta 1992b; 1101:345–352
    [Google Scholar]
  8. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 1984; 12:387–395
    [Google Scholar]
  9. Feinberg A.P., Vogelstein B. A technique for radio-labelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983; 132:6–13
    [Google Scholar]
  10. Fisher R.R., Guillory R.J. Resolution of enzymes catalyzing energy-linked transhydrogenation interaction of trans-hydrogenase factor with the Rhodospirillum rubrum chromatophore membrane. J Biol Chem 1971a; 246:4679–4686
    [Google Scholar]
  11. Fisher R.R., Guillory R.J. Resolution of enzymes catalyzing energy-linked transhydrogenation -preparation and properties of Rhodospirillum rubrum transhydrogenase factor. J Biol Chem 1971b; 246:4687–4693
    [Google Scholar]
  12. Fitzmaurice W.P., Saari L.L., Lowery R.G., Ludden P.W., Roberts G.P. Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodospirillum rubrum. MoI and Gen Genet 1989; 218:340–347
    [Google Scholar]
  13. Glavas N., Ahmad S., Bragg P.D., Olausson T., Rydstrom J. Aspartic acid residues in Escherichia coli transhydrogenase and the exchange of these by site-specific mutagenesis. J Biol Chem 1993; 268:14125–14130
    [Google Scholar]
  14. Harley C.B., Reynolds R.P. Analysis of Escherichia coli promoter sequences. Nucleic Acids Res 1987; 15:2343–2361
    [Google Scholar]
  15. Hatefi Y., Yamaguchi M. Energy-transducing nic-otinamide nucleotide transhydrogenase. In Molecular Mechanisms in Bioenergetics 1992 Edited by Ernster L. Amsterdam: Elsevier; pp 265–281
    [Google Scholar]
  16. Hu P.S., Persson B., Hoog J.O., Jornvall H., Hartog A.F., Berden J.A., Holmberg E., Rydstrom J. Energy-linked transhydrogenase. Characterisation of a nucleotide-binding sequence in nicotinamide nucleotide transhydrogenase from beef heart. Biochim Biophys Acta 1992; 1102:19–29
    [Google Scholar]
  17. Jackson J.B. The proton-translocating nicotinamide adenine dinucleotide transhydrogenase. J Bioenerg Biomembr 1991; 23:715–741
    [Google Scholar]
  18. Kramer R.A., Tomchak L.A., McAndrew S.J., Becker K., Hug D., Pasamontes L., Humbelin M. An Eimeria tenella gene encoding a protein with homology to the nucleotide trans-hydrogenase of Escherichia coli and bovine mitochondria. Mol Biochem Parasitol 1993; 60:327 –332
    [Google Scholar]
  19. Kuroda S., Tanizawa K., Sakamoto Y., Tanaka H., Soda K. Alanine dehydrogenases from two Bacillus species with distinct thermostabilities: molecular cloning, DNA and protein sequence determination and structural comparison with other N AD(P)-dependent dehydrogenases. Biochemistry 1990; 29:1009–1015
    [Google Scholar]
  20. Kustu S., Santero E., Keener J., Topham D., Weiss D. Expression of α-54 (ntr A)-dependent genes is probably united by a common mechanism. Microbiol Rev 1989; 53:367–376
    [Google Scholar]
  21. Kyte J., Doolittle R.F. A simple method for displaying the hydrophobic character of a protein. J Mol Biol 1982; 157:105–132
    [Google Scholar]
  22. Meissner P.S., Sisk W.P., Berman M.L. Bacteriophage lambda cloning system for the construction of directional cDNA libraries. Proc Natl Acad Sci USA 1989; 84:4171–4174
    [Google Scholar]
  23. Olausson T., Hultman T., Holmberg E., Rydstrom J., Ahmad S., Glavas N.A., Bragg P.D. Site-directed mutagenesis of tyrosine residues at nicotinamide nucleotide-binding sites of Escherichia coli transhydrogenase. Biochemistry 1993; 32:13237–13244
    [Google Scholar]
  24. Palmer T., Jackson J.B. Nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus; the H+/H12-ratio and the activation state of the enzyme during reduction of acetyl pyridine adenine dinucleotide. Biochim Biophys Acta 1992; 1099:157–162
    [Google Scholar]
  25. Palmer T., Williams R., Cotton N.P.J., Thomas C.M., Jackson J.B. Inhibition of proton-translocating transhydrogenase from photosynthetic bacteria by N,N-dicyclohexylcarbodiimide. Eur J Biochem 1993; 211:663–669
    [Google Scholar]
  26. Platt T. Transcription termination and the regulation of gene expression. Amu Rev Biochem 1986; 55:339–372
    [Google Scholar]
  27. Rydstrom J., Persson B., Carlenor E. Transhydrogenase linked to pyridine nucleotides. In Pyridine Nucleotide Coenzymes: Chemical Biochemical, and Medical Aspects 1987 Edited by Dolphin D., Poulson R., Avramovic O. New York: John Wiley; 2 pp 433–460
    [Google Scholar]
  28. Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning: a Laboratory Manual 1989 2nd edn Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  29. Sanger F., Nicklen S., Coulson A.R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USsl 1977; 74:5463–5467
    [Google Scholar]
  30. Tong R.C.W., Glavas N.A., Bragg P.D. Topological analysis of the pyridine nucleotide transhydrogenase of E coli using proteolytic enzymes. Biochim Biophys Acta 1991; 1080:19–28
    [Google Scholar]
  31. Von Heijne G. Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol 1992; 225:487–494
    [Google Scholar]
  32. Von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 1986; 14:4683–4690
    [Google Scholar]
  33. Wakabayashi S., Hatefi Y. Amino acid sequence of the NAD(H)-binding region of the mitochondrial nicotinamide nucleotide transhydrogenase modified by N,N'-dicyclohexyl-carbodiimide. Biochem Int 1987a; 15:667–675
    [Google Scholar]
  34. Wakabayashi S., Hatefi Y. Characterization of the substrate-binding sites of the mitochondrial nicotinamide nucleotide transhydrogenase. Biochem Int 1987b; 15:915–924
    [Google Scholar]
  35. Walker J.E. The NADH: ubiquinone oxidoreductase (complex I) of respiratory chains. Q Rev Biophys 1992; 25:253–324
    [Google Scholar]
  36. Weaver P.F., Wall J.D., Gest H. Characterisation of Rhodopseudomonas capsulata. Arch Microbiol 1975; 105:207–216
    [Google Scholar]
  37. Wierenga R.K., Terpstra P., Hoi W.G.J. Prediction of the occurrence of the ADP-binding /J-a-/?-fold in proteins using an amino acid sequence fingerprint. J Membr Biol 1986; 187:101–107
    [Google Scholar]
  38. Yamaguchi M., Hatefi Y. Mitochondrial energy linked nicotinamide nucleotide transhydrogenase. Membrane topography of the bovine enzyme. J Biol Chem 1991; 266:5728–5735
    [Google Scholar]
  39. Yamaguchi M., Hatefi Y. Energy-transducing nicotinamide nucleotide transhydrogenase. Nucleotide-binding properties of the purified enzyme and proteolytic fragments. J Biol Chem 1993; 268:17871–17877
    [Google Scholar]
  40. Yamaguchi M., Hatefi Y., Trach K., Hoch J.A. The primary structure of the mitochondrial energy-linked nicotinamide nucleotide transhydrogenase deduced from the sequence of cDNA clones. J Biol Chem 1988; 263:2761–2767
    [Google Scholar]
  41. Yamaguchi M., Wakabayashi S., Hatefi Y. Mito-chondrial energy-linked nicotinamide nucleotide trans-hydrogenase; effect of substrates on the sensitivity of the enzyme to trypsin and identification of tryptic cleavage sites. Biochemistry 1990; 29:4136–4143
    [Google Scholar]
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