3.D.2 The Proton-translocating Transhydrogenase (PTH) Family
Membrane-associated dimeric proton-translocating, nicotinamide nucleotide transhydrogenases (PTHs; EC 18.104.22.168) catalyze transmembrane translocation of one proton per hydride transferred between NADH and NADP (Jackson et al., 1999; Weston et al., 2002). PTHs are found in some bacteria and in mitochondria from all major groups of eukaryotes. Complete sequences are available from animals, fungi, protozoans and bacteria. Homologues are not present in archaea, in S. cerevisiae and in several bacteria for which complete genomes have been sequenced (Studley et al., 1999). A3-d strcuture is available for a bacterial PTH, and the structures and mechanisms of these proteins have been reviewed (Zhang et al. 2017).
PTH enzymes or enzyme complexes consist of dimers of 1 polypeptide chain (e.g., human PTH), 2 polypeptide chains (e.g., E. coli PTH) or 3 polypeptide chains (e.g., R. rubrum PTH), but all PTH monomers are of about 1000 residues and are homologous throughout their lengths (Hatefi and Yamaguchi, 1996; Yamaguchi and Hatefi, 1994). The animal proteins consist of a hydrophilic N-terminal NAD/NADH-binding domain, a central hydrophobic transmembrane domain of up to 14 transmembrane α-helical spanners, and a C-terminal hydrophilic NADP/NADPH-binding domain. In E. coli, the PTH has been shown to have 13 TMSs, four in the α-subunit and 9 in the β-subunit (Meuller and Rydström, 1999). The C-terminus of the α-subunit and the N-terminus of the β-subunit face the cytosol and periplasm, respectively. The NAD-binding domains are homologous to those of bacterial alanine dehydrogenases and fungal saccharopine dehydrogenases (Studley et al., 1999). The proton-translocation pathway of the PTHs is provided by the central transmembrane domain (Bizouarn et al., 2000, 2002). These enzymes catalyze direct hydride transfer between NAD(H) and NADP(H) in a reaction that is tightly coupled to the transmembrane translocation of a single proton. An imposed proton motive force shifts the equilibrium of the otherwise fully reversible reaction towards NADPH formation (Hatefi and Yamaguchi, 1996; Yamaguchi and Hatefi, 1994). The binding of the 'wrong' nucleotides (NAD+ and NADH instead of NADP+ and NADPH) to the dIII site may lead to slip: proton translocation without change in the nucleotide redox state (Huxley et al., 2011).
3-Dimensional structures of PTH complexed with nucleotide binding components as well as a variety of biochemical and biophysical studies indicate that the dihydronicotinamide ring of NADH can move from a distal position relative to the nicotinamide ring of NADP+ to a proximal position. The movement might be responsible for gating hydride transfer during proton translocation (Cotton et al., 2001; Johansson et al., 2005; Mather et al., 2004; Prasad et al., 1999, 2002; Sedelnikova et al., 2000; Sundaresan et al., 2003; 2005; White et al., 2000). Many mechanistic features are established (Bizouarn et al., 2002; Jackson et al., 2002; Pinheiro et al., 2001; van Boxel et al., 2003). X-ray structural analyses suggest that the two catalytic sites alternate during catalysis. Mutations that inhibit dimerization inhibit NADH binding and greatly lower catalytic activity (Obiozo et al., 2007).
Three invariant amino acids, Arg-127, Asp-135, and Ser-138, in the NAD(H)-binding site of Rhodospirillum rubrum transhydrogenase have been mutated. In each mutant, turnover by the intact enzyme was strongly inhibited. Stopped-flow experiments showed that inhibition results from a block in the steps associated with hydride transfer. Mutation of Asp-135 and Ser-138 had no effect on the binding affinity of either NAD+ or NADH, but mutation of Arg-127 led to much weaker binding of NADH and slightly weaker binding of NAD+. X-ray structures showed that their effects were restricted to the locality of the bound NAD(H). The results were consistent with the suggestion that in the wild-type protein, movement of the Arg-127 side chain, and its hydrogen bonding to Asp-135 and Ser-138, stabilizes the dihydronicotinamide of NADH in the proximal position for hydride transfer (Brondijk et al., 2006; Pedersen et al., 2008).
NADPH/NADP+ homeostasis is critical for countering oxidative stress in cells, and nicotinamide nucleotide transhydrogenase (TH) couples the pmf to the generation of NADPH. Leung et al. 2015 presented the 2.8 Å crystal structure of the transmembrane proton channel domain of TH from Thermus thermophilus (TC# 3.D.2.2.2), and the 6.9 Å crystal structure of the entire enzyme (holo-TH). The membrane domain crystallized as a symmetrical dimer, with each protomer containing a putative proton channel. The holo-TH is a highly asymmetric dimer with the NADPH-binding domain (dIII) in two different orientations. This unusual arrangement suggests a catalytic mechanism in which the two copies of dIII alternatively function in proton translocation and hydride transfer (Leung et al. 2015).
The generalized transport reaction catalyzed by members of the PTH family is:
NADP+ + NADH + H+ (out) NADPH + NAD+ + H+ (in).