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

Voordouw, Gerrit; Vies, Saskia van der; Themmen, Axel P. N.

doi:10.1111/j.1432-1033.1983.tb07293.x

Cited by 45 publications

(37 citation statements)

References 30 publications

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“…At this time we can not exclude the possibility that NADPH could also be oxidized by complex I or that a transhydrogenase could shuffle the electrons from NADPH to NAD 1 before the transfer to complex I. Such a transhydrogenase was found for instance in Pseudomonas fluorescens [19] and also in other organisms [20].…”

Section: Discussionmentioning

confidence: 99%

Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1

Hagemeier

Chistoserdova

Lidstrom

et al. 2000

European Journal of Biochemistry

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Cell extracts of Methylobacterium extorquens AM1 were recently found to catalyze the dehydrogenation of methylene tetrahydromethanopterin (methylene H 4 MPT) with NAD 1 and NADP 1. The purification of a 32-kDa NADP-specific methylene H 4 MPT dehydrogenase (MtdA) was described already. Here we report on the characterization of a second methylene H 4 MPT dehydrogenase (MtdB) from this aerobic a-proteobacterium. Purified MtdB with an apparent molecular mass of 32 kDa was shown to catalyze the oxidation of methylene H 4 MPT to methenyl H 4 MPT with NAD 1 and NADP 1 via a ternary complex catalytic mechanism. The K m for methylene H 4 MPT was 50 mm with NAD 1 (V max 1100 U´mg 21) and 100 mm with NADP 1 (V max 950 U´mg 21 ). The K m value for NAD 1 was 200 mm and for NADP 1 20 mm. In contrast to MtdA, MtdB could not catalyze the dehydrogenation of methylene tetrahydrofolate. Via the N-terminal aminoacid sequence, the MtdB encoding gene was identified to be orfX located in a cluster of genes whose translated products show high sequence identities to enzymes previously found only in methanogenic and sulfate reducing archaea. Despite its location, MtdB did not show sequence similarity to archaeal enzymes. The highest similarity was to MtdA, whose encoding gene is located outside of the archaeal island. Mutants defective in MtdB were unable to grow on methanol and showed a pronounced sensitivity towards formaldehyde. On the basis of the mutant phenotype and of the kinetic properties, possible functions of MtdB and MtdA are discussed. We also report that both MtdB and MtdA can be heterologously overproduced in Escherichia coli making these two enzymes readily available for structural analysis.Keywords: C 1 -metabolism; methylotrophic bacteria; methanogenic archaea; tetrahydromethanopterin; tetrahydrofolate.The methylotrophic a-proteobacterium Methylobacterium extorquens AM1 possesses tetrahydrofolate (H 4 F) and dephospho tetrahydromethanopterin (H 4 MPT), two structural analogue C 1 carriers [1,2]. In cell extracts of this aerobic a-proteobacterium H 4 MPT-and H 4 F-dependent enzymes were found. It was proposed that they participate in two separate C 1 pathways, the one with the H 4 MPT-dependent enzymes being mainly involved in formaldehyde oxidation to CO 2 [1,2].Three [2]. MchA and FfsA were found to have high sequence similarity to the respective enzymes from methanogenic and sulfate reducing archaea and their encoding genes were shown to be located within a cluster of genes whose deduced gene products show, with an exception of OrfX, high sequence similarity to proteins found until now only in methanogenic and sulfate reducing archaea [1]. The gene encoding MtdA is not located within this gene cluster and MtdA does not show significant sequence similarity at the protein level to archaea specific F 420 -dependent or H 2 -forming methylene H 4 MPT dehydrogenases [4]. MtdA shows only minor sequence identity (, 15%) to methylene H 4 F dehydrogenases from bacteria and eucarya [5±9], which is consistent with the fact that ...

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Section: Discussionmentioning

confidence: 99%

Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1

Hagemeier

Chistoserdova

Lidstrom

et al. 2000

European Journal of Biochemistry

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“…bacteria [117]. Thanks to the proton gradient generated across both the mitochondrial inner membrane and the cell membrane of bacteria, the apparent equilibrium constant of the system is changed, causing a shift towards the production of NADPH and NAD + [118].…”

Section: Ec 151: Oxidoreductases Of Chenh Groupsmentioning

confidence: 99%

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

Vidal

Kelly

Mordaka

et al. 2018

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

251

147

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A B S T R A C T NAD(P)H-dependent oxidoreductases catalyze the reduction or oxidation of a substrate coupled to the oxidation or reduction, respectively, of a nicotinamide adenine dinucleotide cofactor NAD(P)H or NAD(P) + . NAD(P)Hdependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilization, the synthesis of chemicals, biodegradation and detoxification. There is great interest in tailoring NAD(P)H-dependent oxidoreductases to make them more suitable for particular applications. Here, we review the main properties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their modification and application.

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“…Both Liang and Houghton [6] and Voordouw et al [15] have suggested that a 94000-100000-M, protein is a component of the E. coli transhydrogenase. Prior to cloning the pnt gene, we attempted to purify the transhydrogenase of E. coli wild-type strain W6 using a method that involved stripping the membrane vesicles with urea, solubilizing the enzyme with deoxycholate in the presence of KCl, followed by ion-exchange chromatography and affinity chromatography using NAD linked to an agarose resin ( Table 2).…”

Section: Purification Of Transhydrogenase From E Coli Strain Jm83 P mentioning

confidence: 99%

Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli

Clarke

Bragg

1985

European Journal of Biochemistry

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1. The nicotinamide nucleotide transhydrogenase of Escherichia coli has been purified from cytoplasmic membranes by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The purified enzyme consists of two subunits, CI and j, of apparent M , 50000 and 47000.2. During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the purified enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient is established across the membrane as indicated by the quenching of the fluorescence of 9-aminoacridine.3. Treatment of transhydrogenase with N,N'-dicyclohexylcarbodiimide results in an inhibition of proton pump activity and transhydrogenation, suggesting that proton translocation and catalytic activities are obligatory linked. NADH protected the enzyme against inhibition by N,N'-dicyclohexylcarbodiimide, while NADP, and to a lesser extent NADPH, appeared to increase the rate of inhibition. [ ''C]Dicyclohexylcarbodiimide preferentially labelled the 50 000-M, subunit of the transhydrogenase enzyme.4. The presence of an allosteric binding site which reacts with NADH, but not with reduced 3-acetylpyridine adenine dinucleotide, has been demonstrated.Pyridine nucleotide transhydrogenase, found in the cytoplasmic membrane of Escherichia coli and in the inner membrane of mitochondria, catalyzes the reversible transfer of a hydride ion equivalent between NAD and NADP. The transhydrogenation between NADH and NADP is coupled to respiration and ATP hydrolysis (see [I] for review).During the past decade, a large body of evidence has accumulated from studies on the mitochondria1 transhydrogenase to support Mitchell's hypothesis [2] that the transhydrogenase functions as a proton pump and translocates protons across the membrane according to the equation [31: nH; + NADPH + NAD nH&, + NADP + NADH .Comparatively little work has been done with the corresponding enzyme from E. coli despite advantages that genetic manipulation of this system can offer. The elucidation of the subunit composition and molecular mechanism of the E. coli transhydrogenase requires purification and reconstitution of the functional enzyme. So far the E. coli transhydrogenase has been only partially purified [4-61. SDS/ polyacrylamide gels of the preparation purified by Liang andCorrespondence to

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

Cited by 45 publications

References 30 publications

Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1

Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli

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