Mapping of Residues in the NADP(H)-binding Site of Proton-translocating Nicotinamide Nucleotide Transhydrogenase fromEscherichia coli

Abstract

Conformational changes in proton pumping transhydrogenases have been suggested to be dependent on binding of NADP(H) and the redox state of this substrate. Based on a detailed amino acid sequence analysis, it is argued that a classical ␤␣␤␣␤ dinucleotide binding fold is responsible for binding NADP(H). A model defining ␤A, ␣B, ␤B, ␤D, and ␤E of this domain is presented. To test this model, four single cysteine mutants (cf␤A348C, cf␤A390C, cf␤K424C, and cf␤R425C) were introduced into a functional cysteine-free transhydrogenase. Also, five cysteine mutants were constructed in the isolated domain III of Escherichia coli transhydrogenase (ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392C, and ecIIIK424C). In addition to kinetic characterizations, effects of sulfhydryl-specific labeling with N-ethylmaleimide, 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid, and diazotized 3-aminopyridine adenine dinucleotide (phosphate) were examined.The Membrane-bound transhydrogenase is composed of three domains. In the Escherichia coli enzyme, domain I (ecI, 1 ␣1 to ϳ␣404) and domain III (ecIII, ϳ␤260 to ␤462) are exposed to the cytosol and contain the binding sites for NAD(H) and NADP(H), respectively. Domain II (ϳ␣405 to ␣510 and ␤1 to ϳ␤260) spans the membrane. Domain I (dI) from E. coli (4, 5), Rhodospirillum rubrum (rrI) (6), and bovine (7), and domain III (dIII) from E. coli (5), R. rubrum (8, 9), and bovine (7, 9) have been overexpressed, purified, and partially characterized. So far, domain II has not been expressed as a separate entity. Interestingly, dII is not required for transhydrogenation to occur (decoupled from proton translocation), as initially shown by Yamaguchi and Hatefi (7). Mixtures of recombinant dI and dIII from the same species or from different species catalyze decoupled "forward" and "reverse" reactions (cf. Reaction 1) and the so-called "cyclic reaction" (which involves the reduction of bound NADP ϩ by NADH, followed by the oxidation of bound NADPH by AcPyAD ϩ ) (5,8,9). From a recent study, it was observed that mixtures of rrI plus rrIII and rrI plus ecIII behaved similarly (10). They catalyzed high cyclic reaction rates (about the same as those observed in the complete E. coli and R. rubrum enzymes) that were limited by the transfer of hydride equivalents (10) and slow reverse reaction rates that, with excess rrI under usual assay conditions, were limited by the release of NADP ϩ (5, 8). With this knowledge at hand, it is now possible to use the rrI plus ecIII system to complement mutagenesis experiments performed on the complete enzyme. In addition to substrate binding affinities and hydride equivalent transfer rates, release rates of NADP ϩ and relative affinities between domains are properties that can be studied in mixtures of rrI and ecIII.A three-dimensional model of the NAD(H)-binding site in E.

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“…Further, the B-face is more exposed to solvent, consistent with biochemical data requiring direct hydride ion transfer between the 4A position of NAD(H) and the 4B position of NADP(H) 1,2 . Chemical modification 10,11 and site-directed mutagenesis data [12][13][14][15][16][17] are in agreement with the observed binding of NADP. Based on these mutagenesis studies, and homology of the E. coli NADP(H) binding domain with classical nucleotide binding folds, a model has been proposed for the structure of the E. coli domain with bound NADP(H) 17 .…”

“…Chemical modification 10,11 and site-directed mutagenesis data [12][13][14][15][16][17] are in agreement with the observed binding of NADP. Based on these mutagenesis studies, and homology of the E. coli NADP(H) binding domain with classical nucleotide binding folds, a model has been proposed for the structure of the E. coli domain with bound NADP(H) 17 . However, in view of the crystal structure of domain III, and the roles of conserved residues, it is unlikely that this model is correct.…”

Critical Role of Water Molecules in Proton Translocation by the Membrane-Bound Transhydrogenase

“…The current structure is of the TH isolated from T. thermophilus , whereas most of the biochemical characterization has been performed with the enzyme from E. coli (Bragg and Hou, 2001; Fjellstrom et al, 1999; Hu et al, 1995; Meuller and Rydstrom, 1999). In comparison, T. thermophilus TH has 924 amino acid residues divided among 3 polypeptide chains (α 1 , α 2 , β) containing 12 transmembrane helices, the E. coli TH has 972 residues divided between 2 polypeptide chains (α and β) with 13 transmembrane helices.…”

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

“…The protruding feature designated helix D/loop D is conformationally mobile [28], and along with the interacting loop E, is thought to have a central role in the transhydrogenase energy-coupling mechanism, perhaps in the crucial transition between the open and occluded states of the enzyme (see below). Mutations of EcbK424, EcbR425 and EcbY431 in helix E and of EcbD392 in helix D/loop D in the intact E. coli enzyme lead to inhibition of transhydrogenation activity [29][30][31][32]. Studies on these and other mutants in loop E and helix D/loop D of isolated E. coli dIII reveal further consequences of interactions of the protein with NADP + and NADPH [33,34].…”