Pyridine Nucleotide Transhydrogenase from <i>Azotobacter vinelandii</i>

Voordouw, Gerrit; Vies, Saskia M. van der; Eweg, J.K.; Veeger, C.; Breemen, J.F.L. van; Bruggen, E.F.J. Van

doi:10.1111/j.1432-1033.1980.tb04948.x

Cited by 14 publications

(21 citation statements)

References 19 publications

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“…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).…”

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confidence: 86%

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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confidence: 86%

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

et al. 1997

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“…In agreement with this finding differ- Following dodecylsulphate gel electrophoresis, the Western blotting procedure was carried out using antibodies against the energy-linked transhydrogenase from beef heart mitochondria ences in amino acid composition for the enzymes from A . viizelandii and P. uerugiizosa have been reported [8]. DiRercnces in amino acid sequence and loss of antigenic determinants are coupled and cause a lower degree of immunological cross-reactivity of functionally related polypeptide chains.…”

Section: And Western Blottingmentioning

confidence: 99%

“…The non-energy-linked pyridine nucleotide transhydrogenase from A. vinelandii was purified as described elsewhere [8] with affinity chromatography using Ado-(2',5')P2 -Sepharose 4B followed by gel filtration through Sephadex G-200. Transhydrogenase from P. aeruginosa was purified using the same procedure (see also [9]).…”

Section: Purification Of Enzymesmentioning

confidence: 99%

Why Are Two Different Types of Pyridine Nucleotide Transhydrogenase Found in Living Organisms?

1983

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Two types of pyridine nucleotide transhydrogenases have been reported in living organisms. The energylinked transhydrogenase is found in mitochondria and in certain heterotrophic and photosynthesizing bacteria, while the non-energy-linkcd transhydrogenase is found in certain heterotrophic bacteria. The presence of a structurally similar non-energy-linked transhydrogenase in A-otohacter vinelandii, Pseudomonas aeruginosa and Pseudomoizas fluorescen.r is readily shown in extrach Tram these bacteria with Western (protein) blotting. This non-energy-linked enzyme is lacking in E.scherichia coli, while the presence of a structurally similar energy-linked enzyme in E. coli and in beef heart mitochondria is indicated with the Western blotting technique. Spinach (Spinacia oleracra) lacks the non-energy-linked transhydrogenase occurring in bacteria. The chloroplast enzyme ferredoxin: NADP+ oxidoreductase, which exhibits non-energy-linked transhydrogenase activity, is immunologically distinct from the bacterial transhydrogenases.In order to provide a rationale for the distribution of the two types of pyridine nucleotide transhydrogenases, the steady-state degrees of reduction of the NADP(H) and NAD(H) pools in A . vinelandii ( R~A D~( H ) and R h A D ( H ) ) have been measured for cells metabolizing sucrose at a variable oxygen flux (&I2). under steadystate conditions explains the energy requirement in the latter reaction. It is found that the degree of reduction of the NADP(H) pool is always higher than that of the NAD(H) poo! ( R~A D~( H ) > RhaucH,) except when 402 goes to zero (R;VADI'(H) z R~A D ( " ) ) . Comparison of these results with literature values indicates that the inequality R h A D P ( H )>The dependence of the non-energy-linked transhydrogenase activity of ferredoxin : NADP' oxidoreductase on RhADp(,{) is compared with that of A . vinelandiii transhydrogenase. The results indicate that this activity is unlikely to be of physiological importance in plant chloroplasts.

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“…Here the folding of polypeptide chains is followed by association to a complete quaternary structure which is the only enzymatically active species. In our own laboratory some protein systems of even greater complexity are currently under investigation like the pyruvate dehydrogenase complex [21] or the flavoprotein pyridine nucleotide transhydrogenase [5].…”

Section: Discussionmentioning

confidence: 99%

“…A . vinelandii cells (130 g) were broken and a cell-free extract obtained similarly as described for the purification of transhydrogenase [5]. This cell-free extract (volume 250 ml, protein concentration 50 mg/ml, isocitrate dehydrogenase activity: 2 Ujmg) was diluted fivefold with 50 mM Tris/HCl pH4 = 8.0, and brought to 50 saturation with ammonium sulphate at 4 "C. After centrifugation the supernatant was brought to 75 "/, saturation and centrifuged.…”

Section: Fur $Cation O F Isocitrale Dehydrogenasementioning

confidence: 99%

Domain Structure of Isocitrate Dehydrogenase from Azotobacter vinelandii

Slobbe

Voordouw

1981

European Journal of Biochemistry

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I . Isocitrate dehydrogenase from Azotobacter vinelundii, which consists of a single polypeptide chain of M , 80000, is completely unfolded in 3 M guanidine hydrochloride at pH 7.5 and 22°C as judged by hydrodynamic criteria and a study of tryptophan fluorescence.2. Transfer of denatured isocitrate dehydrogenase to native solution conditions (less than 0.1 M guanidine hydrochloride) leads to rapid reactivation (zliz z 1 min at 22 "C, pH 7.5). Practically complete reactivation (85 -95 %) can be achieved under properly chosen conditions.3. At high protein concentration (c > 0.5 -1 .0 mg/ml) intermolecular aggregation competes with proper folding and reactivation. Renatured isocitrate dehydrogenase is indistinguishable from the original native enzyme provided that aggregated material is removed by centrifugation and gel filtration. 5. Protease digestion studies using thermolysin or trypsin in 0.4 M guanidine hydrochloride suggest that isocitrate dehydrogenase from A . vinelandii is composed of three domains which, according to their molecular masses, are designated as the 45-kDa, the 19-kDa and the 16-kDa domains. The 45-kDa and 16-kDa domains are resistant to protease digestion in 0.4 M guanidine hydrochloride, while the 19-kDa domain is rapidly digested under these conditions. 6. The (un)folding intermediate D1 thus represents a conformation comprising folded 45-kDa and 16-kDa domains and a largely unfolded 19-kDa domain. In D 2 all three domains have unfolded. The observation that intermolecular aggregation is favored by low concentrations of guanidine hydrochloride (0.3 -0.5 M) indicates that this process may depend on intermolecular interactions between the 45-kDa and 36-kDa domains.

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Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii

Cited by 14 publications

References 19 publications

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

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

Why Are Two Different Types of Pyridine Nucleotide Transhydrogenase Found in Living Organisms?

Domain Structure of Isocitrate Dehydrogenase from Azotobacter vinelandii

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