Membrane-Bound Pyridine Dinucleotide Transhydrogenases

Fisher, Ronald R.; Earle, Steven R.

doi:10.1016/b978-0-12-244750-1.50018-x

Cited by 30 publications

(4 citation statements)

References 117 publications

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“…The AB transhydrogenases specifically transfer the 4A hydrogen of the nicotinamide ring of NADH to NADP ϩ and the 4B hydrogen from NADPH to NAD ϩ , while the BB transhydrogenases transfer the 4B hydrogen from both NADPH and NADH (16). The AB transhydrogenases are found in mitochondria and in some bacteria, such as Escherichia coli (6). They are integral membrane proteins bound to the inner mitochondrial membrane or the bacterial membrane where they couple the transfer of hydride from NADH to NADP ϩ with proton import (12).…”

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The udhA Gene of Escherichia coli Encodes a Soluble Pyridine Nucleotide Transhydrogenase

et al. 1999

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The udhA gene of Escherichia coli was cloned and expressed in E. coli and found to encode an enzyme with soluble pyridine nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a dinucleotide binding domain, not present in published E. coli genome sequences due to a sequencing error. E. coli is hereby the first organism reported to possess both a soluble and a membrane-bound pyridine nucleotide transhydrogenase.

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The udhA Gene of Escherichia coli Encodes a Soluble Pyridine Nucleotide Transhydrogenase

et al. 1999

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“…A membrane-bound, proton-pumping transhydrogenase, specific for the 4A proton of NADH and the 4B proton of NADPH, occurs in mitochondria and in some bacteria, such as Escherichia coli, and has been studied in some detail (3,8). This enzyme couples proton import with oxidation of NADH and reduction of NADP ϩ , and its physiological role is believed to be production of NADPH for reductive biosyntheses.…”

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Cloning, sequence, and properties of the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens

et al. 1997

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The gene encoding the soluble pyridine nucleotide transhydrogenase (STH) of Pseudomonas fluorescens was cloned and expressed in Escherichia coli. STH is related to the flavoprotein disulfide oxidoreductases but lacks one of the conserved redox-active cysteine residues. The gene is highly similar to an E. coli gene of unknown function.Pyridine nucleotide transhydrogenases catalyze the transfer of reducing equivalents between NAD and NADP pools. A membrane-bound, proton-pumping transhydrogenase, specific for the 4A proton of NADH and the 4B proton of NADPH, occurs in mitochondria and in some bacteria, such as Escherichia coli, and has been studied in some detail (3,8). This enzyme couples proton import with oxidation of NADH and reduction of NADP ϩ , and its physiological role is believed to be production of NADPH for reductive biosyntheses. Less wellknown is a soluble transhydrogenase (STH) reported to occur in Pseudomonas fluorescens, Pseudomonas aeruginosa, and Azotobacter vinelandii (13). This enzyme is a flavoprotein specific for the 4B protons of both NADH and NADPH. STH is not energy dependent but is strongly inhibited by NADP ϩ , suggesting that its physiological role is the conversion of NADPH generated by peripheral catabolic pathways in these bacteria to NADH, which can be oxidized for energy generation (16). STH is remarkable for its formation of large polymers. The subunit M r is approximately 54,000, whereas the minimal active form of the P. aeruginosa enzyme has an M r of approximately 1.6 million (18). This minimal form further aggregates on isolation to form filaments of lengths exceeding 500 nm (9). The enzyme from A. vinelandii displays similar behavior, although the structure of the filaments appears to be different (15). STH also shows interesting kinetic behavior, with activity strongly activated by NADPH and 2Ј-AMP and inhibited by NADP ϩ (19). The presence of Ca 2ϩ favors activation and reduces inhibition.To gain some insight into the structural basis for the aggregation and regulation of this unusual enzyme, we sought to clone the gene encoding STH from P. fluorescens NCIMB9815, a close relative of P. aeruginosa.Purification of STH from P. fluorescens. STH activity was assayed by following the reduction of thionicotinamide adenine dinucleotide (tNAD ϩ ) at 400 nm in a reaction mixture consisting of 0.1 mM NADPH and 0.1 mM tNAD ϩ (Sigma Chemical Co.) in 50 mM Tris-HCl buffer (pH 7.0). The molar extinction coefficient of tNADH at 400 nm was taken as 11,300 liters mol Ϫ1 cm Ϫ1 (2). One unit of enzyme activity was defined as that amount of activity reducing 1 mol of tNAD ϩ per min under these conditions. STH was purified from cells of P. fluorescens NCIMB9815 according to a modification of the method of Höjeberg et al.. Cells were grown to stationary phase in 1 liter of SOB medium (14). The cells were harvested by centrifugation (5,000 ϫ g for 15 min) and resuspended in 20 ml of buffer A (50 mM Tris-HCl [pH 7.0] with 2 mM dithiothreitol). The cells were then disrupted by sonication (25 bursts ...

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“…Thus, the 1 5 kD peptide may be associated with a genuine transhydrogenase activity. These results may indicate that the native potato transhydrogenase resembles the mammalian transhydrogenase (4,21,22,24).…”

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confidence: 79%

On the Presence of a Nicotinamide Nucleotide Transhydrogenase in Mitochondria from Potato Tuber

Carlenor

Persson²,

Glaser³

et al. 1988

Plant Physiol.

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Mitochondria isolated from potato (Solanum tuberosum L.) tuber were investigated for the presence of a nicotinamide nucleotide transhydrogenase activity. Submitochondrial particles derived from these mitochondria by sonication catalyzed a reduction of NAD' or 3-acetylpyridine-NAD' by NADPH, which showed a maximum of about 50 to 150 nanomoles/ minute. milligram protein at pH 5 to 6. The Km values for 3-acetylpyridine-NAD' and NADPH were about 24 and 55 micromolar, respectively. Intact mitochondria showed a negligible activity in the absence of detergents. However, in the presence of detergents the specific activity approached about 30% of that seen with submitochondrial particles. The potato mitochondria transhydrogenase activity was sensitive to trypsin and phenylarsine oxide, both agents that are known to inhibit the mammalian transhydrogenase. Antibodies raised against rat liver transhydrogenase crossreacted with two peptides in potato tuber mitochondrial membranes with a molecular mass of 100 to 115 kilodaltons. The observed transhydrogenase activities may be due to an unspecific activity of dehydrogenases and/or to a genuine transhydrogenase. The activity contributions by NADH dehydrogenases and transhydrogenase to the total transhydrogenase activity were investigated by determining their relative sensitivities to trypsin. It is concluded that, at high or neutral pH, the observed transhydrogenase activity in potato tuber submitochondrial particles is due to the presence of a genuine and specific high molecular weight transhydrogenase. At low pH an unspecific reaction of an NADH dehydrogenase with NADPH contributes to the total transhydrogenase activity.active form of the mammalian transhydrogenase is a dimer. The enzyme from beef heart has been extensively investigated with respect to structure and function, especially its mechanism of proton pumping and interaction with other proton pumps in the native membrane as well as in reconstituted liposomes (for reviews, see Refs. 4,21,22,and 24). Transhydrogenase isolated from Escherichia coli has been sequenced and shown to be composed of two subunits of about 50,000 with altogether 11 possible membrane spanning sequences, but otherwise has properties that resemble those of the mammalian enzyme (2, 3).The presence of a low transhydrogenase activity in plants, approximately 10 nmol/min -mg, has been reported previously in mitochondria from pea stems (8, 20) and mung bean (26). In the latter case, an ATP-driven reduction of NADP+ by NADH was also demonstrated (26). However, these activities have been questioned by others, who have explained them as being due to an unspecific NAD(P)H dehydrogenase or by two separate enzyme systems (14, 16). The main reason for the latter conclusion was that no significant NAD+-dependent formation of NADH from NADPH could be demonstrated (14). One possible complication in the measurements may have been that the assay used was based on the natural substrates, which are rapidly oxidized by various dehydrogenases.In the present s...

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Membrane-Bound Pyridine Dinucleotide Transhydrogenases

Cited by 30 publications

References 117 publications

The udhA Gene of Escherichia coli Encodes a Soluble Pyridine Nucleotide Transhydrogenase

The udhA Gene of Escherichia coli Encodes a Soluble Pyridine Nucleotide Transhydrogenase

Cloning, sequence, and properties of the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens

On the Presence of a Nicotinamide Nucleotide Transhydrogenase in Mitochondria from Potato Tuber

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