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. 1991 May 15;88(10):4225–4229. doi: 10.1073/pnas.88.10.4225

cDNA-derived amino acid sequence of the NADH-binding 51-kDa subunit of the bovine respiratory NADH dehydrogenase reveals striking similarities to a bacterial NAD(+)-reducing hydrogenase.

S D Patel 1, R Aebersold 1, G Attardi 1
PMCID: PMC51631  PMID: 2034666

Abstract

A lambda gt10 bovine brain and a lambda gt11 bovine heart cDNA library were screened with oligonucleotide probes corresponding to partial protein sequences directly determined from the isolated 51-kDa subunit of the bovine respiratory-chain NADH dehydrogenase. Clones were isolated that encode a protein of 464 amino acids containing all the 11 partial tryptic peptide sequences determined from the 51-kDa subunit. The size and amino acid composition of this protein agree with those determined for the purified 51-kDa subunit. Furthermore, this protein contains a putative NADH-binding domain, a possible FMN-binding site, and a putative binding site for an iron-sulfur cluster. The above evidence indicates that the cloned protein is the 51-kDa subunit or its precursor. A search for sequence similarity with proteins in the Protein Identification Resource data base has revealed that the 51-kDa subunit has 32% amino acid sequence identity with a major portion of the alpha subunit of the soluble NAD(+)-reducing hydrogenase from Alcaligenes eutrophus. In particular, there are three segments of high sequence similarity (70-88%) between the two proteins which correspond to the three ligand-binding sites.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aebersold R. H., Leavitt J., Saavedra R. A., Hood L. E., Kent S. B. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chen S., Guillory R. J. Studies on the interaction of arylazido-beta-alanyl NAD+ with the mitochondrial NADH dehydrogenase. J Biol Chem. 1981 Aug 25;256(16):8318–8323. [PubMed] [Google Scholar]
  3. Chomyn A., Cleeter M. W., Ragan C. I., Riley M., Doolittle R. F., Attardi G. URF6, last unidentified reading frame of human mtDNA, codes for an NADH dehydrogenase subunit. Science. 1986 Oct 31;234(4776):614–618. doi: 10.1126/science.3764430. [DOI] [PubMed] [Google Scholar]
  4. Chomyn A., Lai S. S. cDNA of the 24 kDa subunit of the bovine respiratory chain NADH dehydrogenase: high sequence conservation in mammals and tissue-specific and growth-dependent expression. Curr Genet. 1989 Aug;16(2):117–126. doi: 10.1007/BF00393404. [DOI] [PubMed] [Google Scholar]
  5. Chomyn A., Mariottini P., Cleeter M. W., Ragan C. I., Matsuno-Yagi A., Hatefi Y., Doolittle R. F., Attardi G. Six unidentified reading frames of human mitochondrial DNA encode components of the respiratory-chain NADH dehydrogenase. Nature. 1985 Apr 18;314(6012):592–597. doi: 10.1038/314592a0. [DOI] [PubMed] [Google Scholar]
  6. Chomyn A., Patel S. D., Cleeter M. W., Ragan C. I., Attardi G. The site of synthesis of the iron-sulfur subunits of the flavoprotein and iron-protein fractions of human NADH dehydrogenase. J Biol Chem. 1988 Nov 5;263(31):16395–16400. [PubMed] [Google Scholar]
  7. Fearnley I. M., Runswick M. J., Walker J. E. A homologue of the nuclear coded 49 kd subunit of bovine mitochondrial NADH-ubiquinone reductase is coded in chloroplast DNA. EMBO J. 1989 Mar;8(3):665–672. doi: 10.1002/j.1460-2075.1989.tb03424.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Galante Y. M., Hatefi Y. Purification and molecular and enzymic properties of mitochondrial NADH dehydrogenase. Arch Biochem Biophys. 1979 Feb;192(2):559–568. doi: 10.1016/0003-9861(79)90126-7. [DOI] [PubMed] [Google Scholar]
  9. HATEFI Y., HAAVIK A. G., GRIFFITHS D. E. Studies on the electron transfer system. XL. Preparation and properties of mitochondrial DPNH-coenzyme Q reductase. J Biol Chem. 1962 May;237:1676–1680. [PubMed] [Google Scholar]
  10. Han A. L., Yagi T., Hatefi Y. Studies on the structure of NADH:ubiquinone oxidoreductase complex: topography of the subunits of the iron-sulfur flavoprotein component. Arch Biochem Biophys. 1988 Dec;267(2):490–496. doi: 10.1016/0003-9861(88)90055-0. [DOI] [PubMed] [Google Scholar]
  11. Hartl F. U., Pfanner N., Nicholson D. W., Neupert W. Mitochondrial protein import. Biochim Biophys Acta. 1989 Jan 18;988(1):1–45. doi: 10.1016/0304-4157(89)90002-6. [DOI] [PubMed] [Google Scholar]
  12. Hatefi Y., Stempel K. E. Isolation and enzymatic properties of the mitochondrial reduced diphosphopyridine nucleotide dehydrogenase. J Biol Chem. 1969 May 10;244(9):2350–2357. [PubMed] [Google Scholar]
  13. Hattori M., Sakaki Y. Dideoxy sequencing method using denatured plasmid templates. Anal Biochem. 1986 Feb 1;152(2):232–238. doi: 10.1016/0003-2697(86)90403-3. [DOI] [PubMed] [Google Scholar]
  14. Ise W., Haiker H., Weiss H. Mitochondrial translation of subunits of the rotenone-sensitive NADH:ubiquinone reductase in Neurospora crassa. EMBO J. 1985 Aug;4(8):2075–2080. doi: 10.1002/j.1460-2075.1985.tb03894.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  16. Ohnishi T., Ragan C. I., Hatefi Y. EPR studies of iron-sulfur clusters in isolated subunits and subfractions of NADH-ubiquinone oxidoreductase. J Biol Chem. 1985 Mar 10;260(5):2782–2788. [PubMed] [Google Scholar]
  17. Ohtsuka E., Matsuki S., Ikehara M., Takahashi Y., Matsubara K. An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions. J Biol Chem. 1985 Mar 10;260(5):2605–2608. [PubMed] [Google Scholar]
  18. Patel S. D., Cleeter M. W., Ragan C. I. Transmembrane organization of mitochondrial NADH dehydrogenase as revealed by radiochemical labelling and cross-linking. Biochem J. 1988 Dec 1;256(2):529–535. doi: 10.1042/bj2560529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pilkington S. J., Walker J. E. Mitochondrial NADH-ubiquinone reductase: complementary DNA sequences of import precursors of the bovine and human 24-kDa subunit. Biochemistry. 1989 Apr 18;28(8):3257–3264. doi: 10.1021/bi00434a021. [DOI] [PubMed] [Google Scholar]
  20. Proudfoot N. J., Brownlee G. G. 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976 Sep 16;263(5574):211–214. doi: 10.1038/263211a0. [DOI] [PubMed] [Google Scholar]
  21. Roise D., Horvath S. J., Tomich J. M., Richards J. H., Schatz G. A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipid bilayers. EMBO J. 1986 Jun;5(6):1327–1334. doi: 10.1002/j.1460-2075.1986.tb04363.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rossmann M. G., Moras D., Olsen K. W. Chemical and biological evolution of nucleotide-binding protein. Nature. 1974 Jul 19;250(463):194–199. doi: 10.1038/250194a0. [DOI] [PubMed] [Google Scholar]
  23. Runswick M. J., Gennis R. B., Fearnley I. M., Walker J. E. Mitochondrial NADH:ubiquinone reductase: complementary DNA sequence of the import precursor of the bovine 75-kDa subunit. Biochemistry. 1989 Nov 28;28(24):9452–9459. doi: 10.1021/bi00450a031. [DOI] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schiffer M., Edmundson A. B. Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys J. 1967 Mar;7(2):121–135. doi: 10.1016/S0006-3495(67)86579-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Scrutton N. S., Berry A., Perham R. N. Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature. 1990 Jan 4;343(6253):38–43. doi: 10.1038/343038a0. [DOI] [PubMed] [Google Scholar]
  27. Tran-Betcke A., Warnecke U., Böcker C., Zaborosch C., Friedrich B. Cloning and nucleotide sequences of the genes for the subunits of NAD-reducing hydrogenase of Alcaligenes eutrophus H16. J Bacteriol. 1990 Jun;172(6):2920–2929. doi: 10.1128/jb.172.6.2920-2929.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wallace R. B., Johnson M. J., Hirose T., Miyake T., Kawashima E. H., Itakura K. The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA. Nucleic Acids Res. 1981 Feb 25;9(4):879–894. doi: 10.1093/nar/9.4.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yagi T., Dinh T. M. Identification of the NADH-binding subunit of NADH-ubiquinone oxidoreductase of Paracoccus denitrificans. Biochemistry. 1990 Jun 12;29(23):5515–5520. doi: 10.1021/bi00475a015. [DOI] [PubMed] [Google Scholar]

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