Mutsuo Yamaguchi

Crystal structure of transhydrogenase domain III at 1.2 Å resolution

The nicotinamide nucleotide transhydrogenases (TH) of mitochondria and bacteria are membrane-intercalated proton pumps that transduce substrate binding energy and protonmotive force via protein conformational changes. In mitochondria, TH utilizes protonmotive force to promote direct hydride ion transfer from NADH to NADP, which are bound at the distinct extramembranous domains I and III, respectively. Domain II is the membrane-intercalated domain and contains the enzymes proton channel. This paper describes the crystal structure of the NADP(H) binding domain III of bovine TH at 1.2 Å resolution. The structure reveals that NADP is bound in a manner inverted from that previously observed for nucleotide binding folds. The non-classical binding mode exposes the NADP(H) nicotinamide ring for direct contact with NAD(H) in domain I, in accord with biochemical data. The surface of domain III surrounding the exposed nicotinamide is comprised of conserved residues presumed to form the interface with domain I during hydride ion transfer. Further, an adjacent region contains a number of acidic residues, forming a surface with negative electrostatic potential which may interact with extramembranous loops of domain II. Together, the distinctive surface features allow mechanistic considerations regarding the NADP(H)-promoted conformation changes that are involved in the interactions of domain III with domains I and II for hydride ion transfer and proton translocation.

Energy-transducing nicotinamide nucleotide transhydrogenase: Nucleotide sequences of the genes and predicted amino acid sequences of the subunits of the enzyme fromRhodospirillum rubrum

Based on the amino acid sequence of the N-terminus of the soluble subunit of theRhodospirillum rubrum nicotinamide nucleotide transhydrogenase, two oligonucleotide primers were synthesized and used to amplify the corresponding DNA segment (110 base pairs) by the polymerase chain reaction. Using this PCR product as a probe, one clone with the insert of 6.4kbp was isolated from a genomic library ofR. rubrum and sequenced. This sequence contained three open reading frames, constituting the genesnntA1, nntA2, andnntB of theR. rubrum transhydrogenase operon. The polypeptides encoded by these genes were designated α1, α2, and β, respectively, and are considered to be the subunits of theR. rubrum transhydrogenase. The predicted amino acid sequence of the α1 subunit (384 residues; molecular weight 40276) has considerable sequence similarity to the α subunit of theEscherichia coli and the N-terminal 43-kDa segment of the bovine transhydrogenases. Like the latter, it has a βαβ fold in the corresponding region, and the purified, soluble α 1 subunit cross-reacts with antibody to the bovine N-terminal 43-kDa fragment. The predicted amino acid sequence of the β subunit of theR. rubrum transhydrogenase (464 residues; molecular weight 47808) has extensive sequence identity with the β subunit of theE. coli and the corresponding C-terminal sequence of the bovine transhydrogenases. The chromatophores ofR. rubrum contain a 48-kDa polypeptide, which cross-reacts with antibody to the C-terminal 20-kDa fragment of the bovine transhydrogenase. The predicted amino acid sequence of the α2 subunit of theR. rubrum enzyme (139 residues; molecular weight 14888) has considerable sequence identity in its C-terminal half to the corresponding segments of the bovine and the α subunit of theE. coli transhydrogenases.

Division of Labor in Transhydrogenase by alternating proton translocation and hydride transfer

Dueling dimers serve dual purposes Both bacteria and mitochrondria produce NADPH for amino acid biosynthesis and to remove reactive oxygen species. The enzyme that makes NADPH must translocate a proton across the membrane and transfer a hydride from NADH to NADP+—processes that happen some 40 Å apart. To understand this complex geometry, Leung et al. solved the structures of the entire transhydrogenase enzyme and the membrane domain from the bacterium Thermus thermophilus (see the Perspective by Krengel and Törnroth-Horsefield). The entire enzyme exists as a dimer, with the two membrane domains in alternate orientations. One of the membrane domains interacts with the membrane component for proton translocation, whereas the other domain exchanges hydride with NAD(H) in another large soluble domain. Science, this issue p. 178; see also p. 125 One monomer in a transmembrane dimer transports protons and the other transfers hydride. [Also see Perspective by Krengel and Törnroth-Horsefield] NADPH/NADP+ (the reduced form of NADP+/nicotinamide adenine dinucleotide phosphate) homeostasis is critical for countering oxidative stress in cells. Nicotinamide nucleotide transhydrogenase (TH), a membrane enzyme present in both bacteria and mitochondria, couples the proton motive force to the generation of NADPH. We present the 2.8 Å crystal structure of the transmembrane proton channel domain of TH from Thermus thermophilus and the 6.9 Å crystal structure of the entire enzyme (holo-TH). The membrane domain crystallized as a symmetric dimer, with each protomer containing a putative proton channel. The holo-TH is a highly asymmetric dimer with the NADP(H)–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.

Chapter 11 The energy-transducing nicotinamide nucleotide transhydrogenase

Publisher Summary This chapter discusses the energy-transducing nicotinamide nucleotide transhydrogenase. The energy-transducing nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are membrane-bound enzymes that catalyze the direct and stereospecific transfer of a hydride ion between the 4A position of NAD (H) and the 4B position of NADP (H). In the mitochondria, the enzyme is embedded in the inner membrane, with its nucleotide binding sites protruding into the matrix, and the transhydrogenation reaction is coupled to transmembrane proton translocation with a H + / H - stoichiometry of unity. Because of the stereospecificity of hydride ion transfer, this type of transhydrogenase is referred to as “AB-transhydrogenase.” The known AB-transhydrogenases are integral membrane proteins, their scalar transhydrogenation reaction is coupled to proton translocation, and they contain separate binding sites for NAD (H) and NADP (H), and have no prosthetic groups. The BB-type transhydrogenases are water-soluble, do not pump protons, are flavoproteins containing FAD, have a single substrate-binding site, and are thought to be concerned with equilibrating the cellular NAD (H) and NADP (H) pools.