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PubMed2003

Conformational change in the NADP(H) binding domain of transhydrogenase defines four states.

Proton-translocating transhydrogenase (TH) couples direct and stereospecific hydride transfer between NAD(H) and NADP(H), bound to soluble domains dI and dIII, respectively, to proton translocation across a membrane bound domain, dII. The reaction occurs with proton-gradient coupled conformational changes, which affect the energetics of substrate binding and interdomain interactions. The crystal structure of TH dIII from Rhodospirillum rubrum has been determined in the presence of NADPH (2.4 A) and NADP (2.1 A) (space group P6(1)22). Each structure has two molecules in the asymmetric unit, differing in the conformation of the NADP(H) binding loop D. In one molecule, loop D has an open conformation, with the B face of (dihydro)nicotinamide exposed to solvent. In the other molecule, loop D adopts a hitherto unobserved closed conformation, resulting in close interactions between NADP(H) and side chains of the highly conserved residues, betaSer405, betaPro406, and betaIle407. The conformational change shields the B face of (dihydro)nicotinamide from solvent, which would block hydride transfer in the intact enzyme. It also alters the environments of invariant residues betaHis346 and betaAsp393. However, there is little difference in either the open or the closed conformation upon change in oxidation state of nicotinamide, i.e., for NADP vs. NADPH. Consequently, the occurrence of two loop D conformations for both substrate oxidation states gives rise to four states: NADP-open, NADP-closed, NADPH-open, and NADPH-closed. Because these states are distinguished by protein conformation and by net charge they may be important in the proton translocating mechanism of intact TH.

Binding Sites↗
PubMed1996

Interaction of nucleotides with the NAD(H)-binding domain of the proton-translocating transhydrogenase of Rhodospirillum rubrum.

Transhydrogenase catalyzes the reduction of NADP+ by NADH coupled to the translocation of protons across a membrane. The polypeptide composition of the enzyme in Rhodospirillum rubrum is unique in that the NAD(H)-binding domain (called Ths) exists as a separate polypeptide. Ths was expressed in Escherichia coli and purified. The binding of nucleotide substrates and analogues to Ths was examined by one-dimensional proton nuclear magnetic resonance (NMR) spectroscopy and by measuring the quenching of fluorescence of its lone Trp residue. NADH and reduced acetylpyridine adenine dinucleotide bound tightly to Ths, whereas NAD+, oxidized acetylpyridine adenine dinucleotide, deamino-NADH, 5'-AMP and adenosine bound less tightly. Reduced nicotinamide mononucleotide, NADPH and 2'-AMP bound only very weakly to Ths. The difference in the binding affinity between NADH and NAD+ indicates that there may be an energy requirement for the transfer of reducing equivalents into this site in the complete enzyme under physiological conditions. Earlier results had revealed a mobile loop at the surface of Ths (Diggle, C., Cotton, N. P. J., Grimley, R. L., Quirk, P. G., Thomas, C. M., and Jackson, J. B. (1995) Eur. J. Biochem. 232, 315-326); the loop loses mobility when Ths binds nucleotide; the reaction involves two steps. This was more clearly evident, even for tight-binding nucleotides, when experiments were carried out at higher temperatures (37 degrees C), where the resonances of the mobile loop were substantially narrower. The binding of adenosine was sufficient to initiate loop closure; the presence of a reduced nicotinamide moiety in the dinucleotide apparently serves to tighten the binding. Two-dimensional 1H NMR spectroscopy of the Ths-5'-AMP complex revealed nuclear Overhauser effect interactions between protons of amino acid residues in the mobile loop (including those in a Tyr residue) and the nucleotide. This suggests that, in the complex, the loop has closed down to within 0.5 nm of the nucleotide.

Biological Transport↗
PubMed1989

Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893.

The mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer Mr 110,000. It is located in the inner mitochondrial membrane and catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner membrane. The amino acid sequence and the nucleotide binding sites of the enzyme have been determined [Yamaguchi, M., Hatefi, Y., Trach, K., & Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767; Wakabayashi, S., & Hatefi, Y. (1987) Biochem. Int. 15, 915-924]. N-Ethylmaleimide, as well as other sulfhydryl group modifiers, inhibits the transhydrogenase. The presence of NADP in the incubation mixture suppressed the inhibition rate by N-ethylmaleimide, and the presence of NADPH greatly increased it. NAD and NADH had little or no effect. The NADPH effect was concentration dependent and saturable, with a half-maximal NADPH concentration effect close to the Km of the enzyme for NADPH. Study of the effect of pH on the N-ethylmaleimide inhibition rate showed that NADPH binding by the enzyme lowers the apparent pKa of the N-ethylmaleimide-sensitive group by 0.4 of a pH unit and NADP binding raises this pKa by 0.4 of a pH unit, thus providing a rationale for the effects of NADP and NADPH on the N-ethylmaleimide inhibition rate. With the use of N-[3H]ethylmaleimide, the modified sulfhydryl group involved in the NADP(H)-modulated inhibition of the transhydrogenase was identified as that belonging to Cys-893, which is located 113 residues upstream of the tyrosyl residue modified by [p-(fluorosulfonyl)benzoyl]-5'-adenosine at the putative NADP(H) binding site of the enzyme (see above references).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Titration of E. coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range.

A pH-titration 2D NMR study of Escherichia coli transhydrogenase domain III with bound NADP(+) or NADPH has been carried out, in which the pH was varied between 5.4 and 12. In this analysis, individual amide protons served as reporter groups. The apparent pK(a) values of the amide protons, determined from the pH-dependent chemical shift changes, were attributed to actual pK(a) values for several titrating residues in the protein. The essential Asp392 is shown to be protonated at neutral pH in both the NADP(+) and NADPH forms of domain III, but with a marked difference in pK(a) not only attributable to the charge difference between the substrates. Titrating residues found in loop D/alpha5 point to a conformational difference of these structural elements that is redox-dependent, but not pH dependent. The observed apparent pK(a) values of these residues are discussed in relation to the crystal structure of Rhodospirillum rubrum domain III, the solution structure of E. coli domain III and the mechanism of intact proton-translocating transhydrogenase.

Escherichia coli Proteins↗
PubMed1995

Site-directed mutagenesis of the redox-active cysteines of Trypanosoma cruzi trypanothione reductase.

The gene for trypanothione reductase from the Silvio strain of Trypanosoma cruzi has been cloned, sequenced and overexpressed in Escherichia coli using the constitutive lpp promoter on the expression plasmid pBSTNAV. Up to 13% of the total soluble protein is enzymically active trypanothione reductase with kinetic properties similar to the enzyme purified from T. cruzi. In order to assess the catalytic role of the putative active-site cysteine residues (C53 and C58), three mutant proteins have been constructed by site-directed mutagenesis substituting alanine or serine residues for cysteine; [C53A]trypanothione reductase, [C53S]trypanothione reductase and [C58S]trypanothione reductase. Although the purified, recombinant mutant proteins were catalytically inactive with NADPH and trypanothione disulphide as substrates, all showed comparable levels of transhydrogenase activity between NADPH and thio-NADP+, suggesting that the mutant proteins had correctly folded in vivo. All three mutants showed substantially different catalytic parameters for thio-NADP+ than the wild-type enzyme, presumably as a consequence of modifying the environment of the enzyme-bound flavin, thereby altering its chemical reactivity. The purified [C58S]trypanothione reductase showed spectral properties similar to the oxidised wild-type enzyme but, unlike the wild-type enzyme, did not acquire the characteristic charge-transfer complex of the EH2 form on addition of NADPH. In contrast, in the absence of NADPH both [C53A]trypanothione reductase and [C53S]trypanothione reductase showed spectral properties similar to the EH2 form of the wild-type enzyme. These data indicate that both C53 and C58 are essential for overall catalysis, with the thiolate anion of C58 interacting with the enzyme-bound FAD and C53 interacting with the disulphide substrate. These mutants should be useful in crystallographic studies of reaction intermediates which cannot be obtained with the catalytically active native enzyme.

Animals↗
PubMed1999

NMR characterization of the NADP(H)-binding domain of Escherichia coli transhydrogenase: sequential assignment and global fold.

The soluble NADP(H)-binding domain of Escherichia coli transhydrogenase (186 amino acids, 20.4 kDa, rotational correlation time 14 ns) was characterized using NMR techniques. The global fold is similar to that of a classical dinucleotide-binding fold with six parallel beta-strands in a central sheet surrounded by helices and irregular structures, but is lacking both alphaD and alphaE. The substrate is bound in an extended conformation at the C-terminal end of the parallel beta-sheet and our data support the notion of a redox dependent structural rearrangement.

Amino Acid Sequence↗
PubMed1992

A mutation at Gly314 of the beta subunit of the Escherichia coli pyridine nucleotide transhydrogenase abolishes activity and affects the NADP(H)-induced conformational change.

Escherichia coli RH1 contains a mutation causing complete loss of pyridine nucleotide transhydrogenase activity. A single base change in the chromosomal DNA resulted in the replacement of Gly314 of the beta subunit by a Glu residue. The mutant enzyme was partially purified and its trypsin cleavage products examined. The distinct pattern of polypeptides given by proteolysis of the normal transhydrogenase in the presence of NADP(H) was absent when the mutant enzyme was treated with trypsin. However, the beta subunit of the mutant enzyme retained its ability to bind to NAD-agarose. Further substitutions were made at Gly314 converting it to Ala, Val or Cys by the use of site-directed mutagenesis. All substitutions for Gly314 abolished the activity completely. The enzyme containing the Gly314----Ala mutation was studied in detail and behaved exactly as the enzyme containing the Gly314----Glu mutation. It is concluded that the mutation in the beta subunit abolished the NADP(H)-induced conformational change in the mutant enzyme. This conformational change, caused by NADP(H) binding, is required to cleave the normal beta subunit at Arg265 by trypsin. The genes encoding the pyridine nucleotide transhydrogenase were completely resequenced and several corrections have been made to the previously published sequence [Clarke et al. (1986) Eur. J. Biochem. 158, 647-653].

Amino Acid Sequence↗
PubMed1997

Mutation of conserved residues in the NADP(H)-binding domain of the proton translocating pyridine nucleotide transhydrogenase of Escherichia coli.

Possible NADP(H)-binding sites of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli were examined by site-directed mutagenesis. The sequence of the beta subunit at positions 314-350 showed several features typical of NADP(H)-binding sites. Mutation of betaGly314, the first glycine residue of the GXGXXV motif, and of betaArg350, which probably interacts with the 2'-phosphate of the substrate NADP(H), resulted in drastic loss of enzyme activity. The loss of activity in the betaArg350 mutants was not due to loss of ability to bind NADP(H). Several residues (betaVal319, betaGly337, betaHis345, and betaArg350) were mutated to make the sequence more similar to that of a NAD(H)-binding site. The introduction of multiple mutations resulted in improper assembly of the enzyme and decreased incorporation into the membrane. The GXGXXG motif, typical of beta alphabeta nucleotide-binding folds, in the sequence of the beta subunit at positions 274-279 was mutated without causing major changes in transhydrogenase activities. It is unlikely to be part of a nucleotide-binding domain. Deletion of the carboxy-terminal 32 amino acids of the beta subunit, a possible nucleotide-binding site, prevented assembly and incorporation of the truncated enzyme into the cytoplasmic membrane of E. coli.

Amino Acid Sequence↗
PubMed1999

A shift in the equilibrium constant at the catalytic site of proton-translocating transhydrogenase: significance for a 'binding-change' mechanism.

In mitochondria and bacteria, transhydrogenase uses the transmembrane proton gradient (Deltap) to drive reduction of NADP+ by NADH. We have investigated the pre-steady-state kinetics of NADP+ reduction by acetylpyridine adenine dinucleotide (AcPdADH, an analogue of NADH) in complexes formed from the two, separately prepared, recombinant, peripheral subunits of the enzyme: the dI component, which binds NAD+ and NADH, and the dIII component, which binds NADP+ and NADPH. In the stopped-flow spectrophotometer the reaction proceeds as a single-turnover burst of hydride transfer to NADP+ on dIII before product NADPH release becomes limiting in steady state. The burst is biphasic. The results indicate that the fast phase represents direct hydride transfer from AcPdADH to NADP+ in dI:dIII complexes, and that the slow phase, which predominates when [dI]<[dIII], corresponds to dissociation of the protein complexes during multiple turnovers of dI. Measurements on the amplitude of the burst, and on the apparent first-order rate constant of the fast phase, indicate that the equilibrium constant of the hydride-transfer step on the enzyme is shifted relative to that in solution. This has consequences for a model proposed earlier, in which Deltap is used, not at the hydride-transfer step, but to change the binding affinities of NADP+ and NADPH.

Catalysis↗
PubMed2004

Conformational diversity in NAD(H) and interacting transhydrogenase nicotinamide nucleotide binding domains.

Transhydrogenase (TH) couples direct and stereospecific hydride transfer between NAD(H) and NADP(H), bound within soluble domains I and III, respectively, to proton translocation across membrane bound domain II. The cocrystal structure of Rhodospirillum rubrum TH domains I and III has been determined in the presence of limiting NADH, under conditions in which the subunits reach equilibrium during crystallization. The crystals contain three heterotrimeric complexes, dI(2)dIII, in the asymmetric unit. Multiple conformations of loops and side-chains, and NAD(H) cofactors, are observed in domain I pertaining to substrate/product exchange, and highlighting electrostatic interactions during the hydride transfer. Two interacting NAD(H)-NADPH pairs are observed where alternate conformations of the NAD(H) phosphodiester and conserved arginine side-chains are correlated. In addition, the stereochemistry of one NAD(H)-NADPH pair approaches that expected for nicotinamide hydride transfer reactions. The cocrystal structure exhibits non-crystallographic symmetry that implies another orientation for domain III, which could occur in dimeric TH. Superposition of the "closed" form of domain III (PDB 1PNO, chain A) onto the dI(2)dIII complex reveals a severe steric conflict of highly conserved loops in domains I and III. This overlap, and the overlap with a 2-fold related domain III, suggests that motions of loop D within domain III and of the entire domain are correlated during turnover. The results support the concept that proton pumping in TH is driven by the difference in binding affinity for oxidized and reduced nicotinamide cofactors, and in the absence of a difference in redox potential, must occur through conformational effects.

Bacterial Proteins↗
PubMed1988

The alternative respiratory pathway of the yeast Candida parapsilosis: oxidation of exogenous NAD(P)H.

The yeast Candida parapsilosis possesses two routes of electron transfer from exogenous NAD(P)H to oxygen. Electrons are transferred either to the classical cytochrome pathway at the level of ubiquinone through an NAD(P)H dehydrogenase, or to an alternative pathway at the level of cytochrome c through another NAD(P)H dehydrogenase which is insensitive to antimycin A. Analyses of mitoplasts obtained by digitonin/osmotic shock treatment of mitochondria purified on a sucrose gradient indicated that the NADH and NADPH dehydrogenases serving the alternative route were located on the mitochondrial inner membrane. The dehydrogenases could be differentiated by their pH optima and their sensitivity to amytal, butanedione and mersalyl. No transhydrogenase activity occurred between the dehydrogenases, although NADH oxidation was inhibited by NADP+ and butanedione. Studies of the effect of NADP+ on NADH oxidation showed that the NADH:ubiquinone oxidoreductase had Michaelis-Menten kinetics and was inhibited by NADP+, whereas the alternative NADH dehydrogenase had allosteric properties (NADH is a negative effector and is displaced from its regulatory site by NAD+ or NADP+).

Candida↗
PubMed1991

The proton-translocating nicotinamide adenine dinucleotide transhydrogenase.

H(+)-transhydrogenase couples the reversible transfer of hydride ion equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. There are separate sites on the enzyme for the binding of NAD(H) and of NADP(H). There are some indications of the position of the binding sites in the primary sequence of the enzymes from mitochondria and Escherichia coli. Transfer of hydride ion equivalents only proceeds when a reduced and an oxidized nucleotide are simultaneously bound to the enzyme. When delta p = 0 the rate of interconversion of the ternary complexes of enzyme and nucleotide substrates is probably limiting. An increase in delta p accelerates the rate of interconversion in the direction of NADH----NADP+ until another kinetic component, possibly product release, becomes limiting. The available data are consistent with either direct or indirect mechanisms of energy coupling.

Animals↗
PubMed1993

Activation and inhibition of mitochondrial transhydrogenase by metal ions.

Mitochondrial transhydrogenase has been reported previously to be inhibited by high, rather non-physiological concentrations (in the range of 2-20 mM) of divalent cations. We show that the enzyme could be activated by low (from about 1 microM to 1 mM) concentrations of Ca2+ and Mg2+, which are within physiological range. These results bring in line the effects observed with mitochondrial enzyme to the findings with bacterial transhydrogenases. The activation of transhydrogenase by divalent cations is interpreted as an increase in affinity of the NADP(H)-binding site of the enzyme-NAD(H) complex. Reported effects of the metal ions could be important for the enzyme function in vivo.

Animals↗