Thioredoxin reduction dependent on α‐ketoacid oxidation by α‐ketoacid dehydrogenase complexes

Bunik, Victoria I.; Follmann, Hartmut

doi:10.1016/0014-5793(93)80801-z

Cited by 38 publications

(32 citation statements)

References 11 publications

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“…Alternatively, it represents specific catalytic properties of the individual proteins. With the insulin assay we could also observe reduction of T. brucei thioredoxin by dihydrolipoamide as reported earlier for E. coli thioredoxin (26,43). However, almost all cellular lipoic acid is amide linked to protein components of mitochondrial multienzyme complexes.…”

Section: Reduction Of T Brucei Thioredoxin By Trypanothione and Othersupporting

confidence: 54%

Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei

Schmidt

Krauth‐Siegel

2003

Journal of Biological Chemistry

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Trypanosoma brucei, the causative agent of African sleeping sickness, possesses a single thioredoxin that has an unusually high pI value of 8.5 and lacks a conserved aspartyl residue claimed to be involved in catalysis in other thioredoxins. Despite these peculiarities, T. brucei thioredoxin behaves like classical thioredoxins. It is reduced by thioredoxin reductases from different species, serves as donor of reducing equivalents for the ribonucleotide reductase of the parasite, and catalyzes the reduction of protein disulfides. The redox potential of ؊267 mV was obtained from protein-protein redox equilibration with Escherichia coli thioredoxin. The pK value of T. brucei thioredoxin was determined by two different methods. Carboxamidomethylation of the reduced protein yielded a pK value of 7.4 and generated mono-alkylated protein. The thiolate absorption at 240 nm resulted in a pK of 7.6 and, based on the extinction coefficient of 11.6 mM ؊1 cm ؊1 , there are two (or three) cysteines titrating with very similar pK values. A thioredoxin reductase has not yet been detected in any organism of the order Kinetoplastida. T. brucei thioredoxin is spontaneously reduced by trypanothione (bis(glutathionyl)spermidine). Obviously, a specific thioredoxin reductase is not required as thioredoxin reduction can be conducted by the parasite-specific trypanothione/ trypanothione reductase system. Trypanosomatids such as Trypanosoma brucei, the causative agent of African sleeping sickness, have a unique thiol metabolism in which the ubiquitous glutathione/glutathione reductase couple is replaced by trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine), and the flavoenzyme trypanothione reductase (1, 2). The parasite dithiol is much more reactive than glutathione. It is a spontaneous reductant of dehydroascorbate as well as the disulfide forms of glutathione and ovothiol (2). It reduces tryparedoxin, a 16-kDa dithiol protein with a CPPC active site motif, which distantly belongs to the thioredoxin protein family (3). The trypanothione/tryparedoxin couple is the donor of reducing equivalents for the detoxication of hydroperoxides catalyzed by a cascade of proteins that are composed of trypanothione reductase, trypanothione, tryparedoxin, and a peroxiredoxin-type tryparedoxin peroxidase. Tryparedoxin is also involved in the reduction of ribonucleotide reductase (4) and glutathione peroxidase-like parasite peroxidases (5, 6). The trypanothione metabolism is essential for the parasite. Down-regulation of trypanothione reductase in bloodstream T. brucei by more than 90% results in growth arrest and hypersensitivity toward hydrogen peroxide as well as the loss of infectivity in a mouse model (7).Besides tryparedoxin, T. brucei possesses a single classical thioredoxin with a M r of 12,000 and the conserved WCGPC catalytic site (8). The parasite protein is unusual in having a calculated pI value of 8.5 instead of the acidic pI found for most thioredoxins and a conserved functional aspartate (Asp-26 in Escherichia coli thioredoxin) (9, 10)...

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Section: Reduction Of T Brucei Thioredoxin By Trypanothione and Othersupporting

confidence: 54%

Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei

Schmidt

Krauth‐Siegel

2003

Journal of Biological Chemistry

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“…The 2-oxoglutarate and pyruvate dehydrogenase complexes were obtained from pig heart according to [10] applying the modifications described in [1].…”

Section: Methodsmentioning

confidence: 99%

“…Because the latter is an intermediate of the 2-oxoacid oxidation occuring in mitochondria, we checked whether the organelle-specific redox transformation of thioredoxin may be coupled to the 2-oxoacid oxidation. Indeed, the E. coli thioredoxin becomes reduced at the expense of 2-oxoacid oxidized by mitochondrial 2-oxoacid dehydrogenase complexes [1]. This occurs through dihydrolipoate formed if NAD ÷, the terminal electron acceptor in the complex-catalyzed reaction, is omitted and replaced by free lipoate.…”

Section: -Oxoacid + (S-s)-iip-e2 E1 ---) Co 2 "~-(Acyl-s-sh)-limentioning

confidence: 99%

“…Coupling 2-oxoacid oxidation to the thioredoxin redox cycle with insulin [1] provides an assay for characterizing the lipoatereductase activity of the 2-oxoacid dehydrogenase complexes. This system was used in the present work to reveal the mechanism of Reaction 4.…”

Section: -Oxoacid + (S-s)-iip-e2 E1 ---) Co 2 "~-(Acyl-s-sh)-limentioning

confidence: 99%

“…Recently we have started the investigation of the interplay between dehydrogenases of 2-oxoacids and thioredoxins [1]. The dehydrogenases are multienzyme mitochondrial complexes known to utilize pyruvate, 2-oxoglutarate or branched chain 2-oxoacids in the sequence of Reactions I-3 [2]:…”

Section: Introductionmentioning

confidence: 99%

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Using lipoate enantiomers and thioredoxin to study the mechanism of the 2‐oxoacid‐dependent dihydrolipoate production by the 2‐oxoacid dehydrogenase complexes

et al. 1995

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The thioredoxin-catalyzed insulin reduction by dihydrolipoate was applied to study the 2-oxoacid:lipoate oxidoreductase activity of 2-oxoacid dehydrogenase complexes. The enzymatic and non-enzymatic mechanisms of the transfer of reducing equivalents from the complexes to free lipoic acid (a-lipoic acid, 6,8-thiooctic acid) were distinguished using the high stereoselectivity of the complex enzymes to the R-enantiomer of lipoate. Unlike these enzymes, thioredoxin from E. coli exibited no stereoselectivity upon reduction with chemically obtained dihydrolipoate. However, coupled to the dihydrolipoate production by the dehydrogenase complexes, the process was essentially sensitive both to the enantiomer used and the dihydrolipoyl dehydrogenase activity of the complexes. These results indicated the involvement of the third complex component, dihydrolipoyl dehydrogenase, in the 2-oxoacid-dependent dihydrolipoate formation. The implication of the investigated reaction for a connection between thioredoxin and the 2-oxoacid dehydrogenase complexes in the mitochondrial metabolism are discussed.

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Lipoic Acid Acquisition and Glutathione Biosynthesis in Apicomplexan Parasites

Storm

Patzewitz

Müller

2011

Apicomplexan Parasites

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Thioredoxin reduction dependent on α‐ketoacid oxidation by α‐ketoacid dehydrogenase complexes

Cited by 38 publications

References 11 publications

Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei

Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei

Using lipoate enantiomers and thioredoxin to study the mechanism of the 2‐oxoacid‐dependent dihydrolipoate production by the 2‐oxoacid dehydrogenase complexes

Lipoic Acid Acquisition and Glutathione Biosynthesis in Apicomplexan Parasites

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