Energy-linked transhydrogenase. Characterization of a nucleotide-binding sequence in nicotinamide nucleotide transhydrogenase from beef heart

Hu, P.; Persson, Bengt L.; Jo, Höög; Jörnvall, Hans; Hartog, Aloysius F.; Berden, J.A.; Holmberg, Erland; Rydström, Jan

doi:10.1016/0005-2728(92)90060-f

Cited by 19 publications

(15 citation statements)

References 29 publications

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“…(e.g., Pro, Gly, and Asn) (Richardson & Richardson, 1989). The prediction of a transmembrane a-helix in the extramembranous N-terminal region of both the bovine and the E. coli transhydrogenases (Tables 2 and 3) was not included, since the assignment of this region as a NADH-binding region with a general /3a/3-fold makes this prediction rather unlikely (Hu et al, 1992;Yamaguchi & Hatefi, 1993).…”

Section: Resultsmentioning

confidence: 99%

Prediction and Site-Specific Mutagenesis of Residues in Transmembrane .alpha.-Helixes of Proton-Pumping Nicotinamide Nucleotide Transhydrogenases from Escherichia coli and Bovine Heart Mitochondria

Holmberg¹,

Olausson²,

Hultman³

et al. 1994

Biochemistry

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Nicotinamide nucleotide transhydrogenase from bovine heart consists of a single polypeptide of 109 kD. The complete gene for this transhydrogenase was constructed, and the protein primary structure was determined from the cDNA. As compared to the previously published sequences of partially overlapping clones, three residues differed: Ala591 (previously Phe), Val777 (previously Glu), and Ala782 (previously Arg). The Escherichia coli transhydrogenase consists of an alpha subunit of 52 kD and a beta subunit of 48 kD. Alignment of the protein primary structure of the bovine trashydrogenase with that of the transhydrogenase from E. coli showed an identity of 52%, indicating similarly folded structures. Prediction of transmembrane-spanning alpha-helices, obtained by applying several prediction algorithms to the primary structures of the revised bovine heart and E. coli transhydrogenases, yielded a model containing 10 transmembrane alpha-helices in both transhydrogenases. In E. coli transhydrogenase, four predicted alpha-helices were located in the alpha subunit and six alpha-helices were located in the beta subunit. Various conserved amino acid residues of the E. coli transhydrogenase located in or close to predicted transmembrane alpha-helixes were replaced by site-specific mutagenesis. Conserved negatively charged residues in predicted transmembrane alpha-helices possibly participating in proton translocation were identified as beta Glu82 (Asp655 in the bovine enzyme) and beta Asp213 (asp787 in the bovine enzyme) located close to the predicted alpha-helices 7 and 9 of the beta subunit. beta Glu82 was replaced by Lys or Gln and beta Asp213 by Asn or His. However, the catalytic as well as the proton pumping activity was retained. In contrast, mutagenesis of the conserved beta His91 residue (His664 in the bovine enzyme) to Ser, Thr, and Cys gave an essentially inactive enzyme. Mutation of alpha His450 (corresponding to His481 in the bovine enzyme) to Thr greatly lowered catalytic activity without abolishing proton pumping. Since no other conserved acidic or basic residues were predicted in transmembrane alpha-helices regardless of the prediction algorithm used, proton translocation by transhydrogenase was concluded to involve a basic rather than an acidic residue. The only conserved cysteine residue, beta Cys260 (Cys834 in the bovine enzyme), located in the predicted alpha-helix 10 of the E. coli transhydrogenase, previously suggested to function as a redox-active dithiol, proved not to be essential, suggesting that redox-active dithiols do not play a role in the mechanism of transhydrogenase.

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Section: Resultsmentioning

confidence: 99%

Prediction and Site-Specific Mutagenesis of Residues in Transmembrane .alpha.-Helixes of Proton-Pumping Nicotinamide Nucleotide Transhydrogenases from Escherichia coli and Bovine Heart Mitochondria

Holmberg¹,

Olausson²,

Hultman³

et al. 1994

Biochemistry

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“…These residues are conserved and correspond to «Tyr226 and 0Tyr431 of the E. coli transhydrogenase. They constitute parts of possible nucleotide-binding folds (Brándén & Tooze, 1991; Hu et al, 1992). That the N-terminal nucleotidebinding region binds one NADH and the C-terminal nucleotide-binding region binds one NADPH per dimer in the bovine transhydrogenase was recently demonstrated by Hatefi and co-workers (Yamaguchi & Hatefi, 1993), strongly suggesting that the previous tentative site localization is correct (Hu et al, 1992).…”

mentioning

confidence: 94%

Site-directed mutagenesis of tyrosine residues at nicotinamide nucleotide binding sites of Escherichia coli transhydrogenase

Olausson

Hultman²,

Holmberg³

et al. 1993

Biochemistry

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Nicotinamide nucleotide transhydrogenase (E.C.1.6.1.1) from Escherichia coli was investigated with respect to the role of specific conserved tyrosine residues of putative substrate-binding regions. The enzyme from E. coli is made up of two subunits, alpha (510 residues) and beta (462 residues). The corresponding enzyme from bovine mitochondria is a single polypeptide (1043 residues) whose N-terminal region corresponds to the alpha subunit and whose C-terminal region corresponds to the beta subunit. Tyrosines 245 and 1006 of the mitochondrial enzyme have been shown to react selectively with 5'-(p-fluorosulfonylbenzoyl)adenosine with inactivation of the enzyme. In E. coli these residues correspond to tyrosine 226 of the alpha subunit and tyrosine 431 of the beta subunit. In addition, tyrosine 315 of the beta subunit is of interest since mutation of an adjacent residue (glycine 314) leads to inactivation [Ahmad, S., Glavas, N. A., & Bragg, P. D. (1992) Eur. J. Biochem. 207, 733-739]. In order to assess the role of the aforementioned conserved tyrosine residues in the mechanism and structure of transhydrogenases, these were replaced by site-specific mutagenesis, using the cloned and overexpressed E. coli transhydrogenase genes [Clarke, D. M., & Bragg, P. D. (1985) J. Bacteriol. 162, 367-373]. Phenylalanine mutants of all three tyrosine residues showed approximately 50% activity or more with regard to catalytic activity assayed as reduction of 3-acetylpyridine-NAD+ by NADPH. These mutants were also active in proton pumping assayed as quenching of 9-methoxy-6-chloro-2-aminoacridine or quinacrine fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…The residues are mapped on to our emerging NMR structure of the dIII protein [16]. The existence of a GXGXXA/V sequence motif in dIII led to the suggestion [17–19]that dIII might have a typical dinucleotide‐binding fold [20]. On this basis, Fjellström et al [21]devised a partial model of the Escherichia coli dIII protein using the secondary structure predictions of the PHD programm [22].…”

Section: Introductionmentioning

confidence: 99%

Structural changes in the recombinant, NADP(H)‐binding component of proton translocating transhydrogenase revealed by NMR spectroscopy

et al. 1999

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Energy-linked transhydrogenase. Characterization of a nucleotide-binding sequence in nicotinamide nucleotide transhydrogenase from beef heart

Cited by 19 publications

References 29 publications

Prediction and Site-Specific Mutagenesis of Residues in Transmembrane .alpha.-Helixes of Proton-Pumping Nicotinamide Nucleotide Transhydrogenases from Escherichia coli and Bovine Heart Mitochondria

Prediction and Site-Specific Mutagenesis of Residues in Transmembrane .alpha.-Helixes of Proton-Pumping Nicotinamide Nucleotide Transhydrogenases from Escherichia coli and Bovine Heart Mitochondria

Site-directed mutagenesis of tyrosine residues at nicotinamide nucleotide binding sites of Escherichia coli transhydrogenase

Structural changes in the recombinant, NADP(H)‐binding component of proton translocating transhydrogenase revealed by NMR spectroscopy

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