Proton Translocating NAD(P)+ Transhydrogenase
Cartoon architecture of the holo-Transhydrogenase dimer showing the three conserved domains.
Identifiers
EC no.7.1.1.1
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Proton-Translocating NAD(P)+ Transhydrogenase (E.C. 7.1.1.1) is an enzyme in that catalyzes the translocation of hydrons that are connected to the redox reaction NADH + NADP+ + H+outside => NAD+ + NADPH + H+inside

EC Number 7.1.1.1

EC 7 – Translocases are a grouping of enzymes with a common function of assisting to move a molecule, typically across a membrane.

EC 7.1 – enzymes that catalyze translocation of hydrons (the positive cations of all hydrogen isotopes e.g. protons, deuterons, tritons)

EC 7.1.1 – enzymes that catalyze translocation of hydrons that have a tie to oxidoreductase reactions

EC 7.1.1.1 – proton-translocating NAD(P)+ transhydrogenase.[1]

Reaction pathway

The reaction that this enzyme catalyzes is

NADH + NADP+ + H+outside => NAD+ + NADPH + H+inside

This redox reaction is a transfer of hydride equivalents from NADH to NADP+ coupled to a translocation of protons across a membrane. NADP+ is reduced to NADPH by NADH, which is oxidized into NAD+. This reduction is tied to the inward translocation of protons across a membrane.[2] This process is not necessarily coupled, but is found coupled in nature.[2]

Known organisms and locations

The transhydrogenase is found in the inner mitochondrial membranes of all vertebrates excluding fish. While it is not found in the yeast Saccharomyces cervisiae, it is found in algae such as Acetabularia acetabulum, as well as in the cytosolic membrane of many bacteria, including Escherichia coli.[3] In E. coli the enzyme helps to provide between 35-45% of cytosolic NADPH under standard growth conditions.[4] E. coli will be our model organism when looking at structure and function.

Description of known crystal structures/active sites

The structure of proton-translocating NADP(+) transhydrogenase has been shown to vary between organisms. The structures of the enzyme share an architecture with 3 known domains, dI, dII, dIII, but differ on exact structure and makeup of these domains.[5] Humans have a single polypeptide chain that forms all three domains. Bacteria are split into two main groups. Some are similar to E. coli, which have two subunits, α and β. Other bacteria have a similar β subunit, while their α subunit is split into two so that the dI domain is its own polypeptide.[6] These three domains form a heterotrimer, with both dI and dIII in the cytosolic space and connected to dII. The heterotrimer forms a dimer, with dI as the dimerization unit

dII

The domain II (dII) is around 400 residues and spans across the membrane forming a channel for protons. It has 13 α-helices comprising its transmembrane domain.[6] Since both ends of the dII domain are on the cytosolic side of the membrane, an even number of helices would make more sense, structurally. The current prediction as to why an odd number of helices exists in dII is that the α and β subunits have another helix that connects their C and N termini.[2] dII also has few conserved residues and shorter loops on the periplasmic side of the transmembrane domain, while the cytosolic side has highly conserved and longer loops.[2]

dI

Domain dI is about 400 residues long and protrudes into the cytosolic space with a binding site for NAD(H). When crystallized in its dimerized form (a homodimer), dI has several subdomains, including the binding site for NAD(H).  Two subdomains in particular, dI1 and dI2 both contain a Rossmann fold, which is made of a 6-stranded parallel β-sheet surrounded by α-helices, and are connected by two longer α-helices.[6] The C termini of the two dI monomers forms the substrate-binding cleft for NAD(H).[6]

dIII

Domain dIII is about 200 residues long and also protrudes into the cytosolic space, where it has a binding site for NADP(H). Similar to dI, dIII comprises a Rossmann fold.[7] When in the complete heterotrimer, dIII is thought to extend into the cleft of dI in order to bring their substrate-binding sites, and their substrates, closer together to allow an easier  transfer of hydride ion from NADH to NADP+.[7] A high affinity for NADP(H) is evidenced by loop E, which acts as a lid to allow tighter bonding of the ligand via hydrogen bonding.[7] There is also a fingerprint motif (Gly-X-Gly-X-X-Ala/Val) that increases specificity of the enzyme to bind to the proper substrate which is highly conserved.[7] The link between dIII and dII is less rigid, and consists of a scattering of smaller polar residues as well as a helix D/loop D region that perpendicularly extends from the Rossmann fold.[7]

Structure tied to function

This enzyme’s structure is tied to its function heavily. The transmembrane domain in dII formed by alpha helices lends itself well to its function as a proton channel across a membrane. The lid and fingerprint motifs used by dIII to increase affinity to NADP(H) are also examples of its structure being tied to function. In addition, the ridge of dIII fitting into the cleft of dI shows that even the structure of the dI/dIII dimer is helpful in moving their two substrates closer together to allow hydride transfer.

Since this enzyme exists in multiple organisms, it is possible to look at multiple orthologs of this enzyme. Their structures are not identical, but most are highly similar, and all share the same architecture and domains.[8] This all shows that the structure of this enzyme is closely connected to its role in reduction of NADP+.

Enzyme functions in the cell

NADPH is used as a reducing agent in many anabolic reactions. Proton translocating NAD(P)+  transhydrogenase is one of the main ways that cells can regenerate NADPH after it is used. In E. coli, this pathway contribute equal amounts of NADPH as the pentose phosphate pathway, and both were the main producers of NADPH under standard growth conditions.[4] 

NADP(H) and NAD(H) are equal and opposite contributors to metabolism, and this enzyme is necessary to keep a balance between the two. It can also function to generate a proton gradient across a membrane, as it is a reversible reaction, and is used as such in certain organisms.

“It can contribute to NADPH formation for bio-synthesis and glutathione reduction, in the regulation of flux through the tricarboxylic acid cycle, and, operating in reverse in some invertebrates, it can transfer reducing power from NADPH to NAD+ to fuel an anaerobic respiratory chain.” – J. Baz Jackson, Sarah J. Peake, and Scott A. White on the role of proton translocating NAD(P)+ transhydrogenase in the cell.[3]

References

  1. ↑ Tipton K. "Enzyme Nomenclature News: Translocases (EC 7): A new EC Class". ExplorEnz: The primary source of the IUBMB enzyme list. Retrieved 2 Oct 2019.
  2. 1 2 3 4 Bizouarn T, Fjellström O, Meuller J, Axelsson M, Bergkvist A, Johansson C, et al. (April 2000). "Proton translocating nicotinamide nucleotide transhydrogenase from E. coli. Mechanism of action deduced from its structural and catalytic properties". Biochimica et Biophysica Acta. 1457 (3): 211–28. doi:10.1016/S0005-2728(00)00103-1. PMID 10773166.
  3. 1 2 Jackson JB, Peake SJ, White SA (December 1999). "Structure and mechanism of proton-translocating transhydrogenase". FEBS Letters. 464 (1–2): 1–8. doi:10.1016/S0014-5793(99)01644-0. PMID 10611473. S2CID 45559290.
  4. 1 2 Sauer U, Canonaco F, Heri S, Perrenoud A, Fischer E (February 2004). "The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli". The Journal of Biological Chemistry. 279 (8): 6613–9. doi:10.1074/jbc.M311657200. PMID 14660605.
  5. ↑ Padayatti PS, Leung JH, Mahinthichaichan P, Tajkhorshid E, Ishchenko A, Cherezov V, et al. (July 2017). "Critical Role of Water Molecules in Proton Translocation by the Membrane-Bound Transhydrogenase". Structure. 25 (7): 1111–1119.e3. doi:10.1016/j.str.2017.05.022. PMC 5524145. PMID 28648609.
  6. 1 2 3 4 Johansson T, Oswald C, Pedersen A, Törnroth S, Okvist M, Karlsson BG, et al. (September 2005). "X-ray structure of domain I of the proton-pumping membrane protein transhydrogenase from Escherichia coli". Journal of Molecular Biology. 352 (2): 299–312. doi:10.1016/J.JMB.2005.07.022. PMID 16083909.
  7. 1 2 3 4 5 Jeeves M, Smith KJ, Quirk PG, Cotton NP, Jackson JB (August 2000). "Solution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from Rhodospirillum rubrum". Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1459 (2–3): 248–57. doi:10.1016/S0005-2728(00)00159-6. PMID 11004437.
  8. ↑ Huxley L, Quirk PG, Cotton NP, White SA, Jackson JB (January 2011). "The specificity of proton-translocating transhydrogenase for nicotinamide nucleotides". Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1807 (1): 85–94. doi:10.1016/J.BBABIO.2010.08.005. PMID 20732298.
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