Thioredoxin elicits a new dihydrolipoamide dehydrogenase activity by interaction with the electron-transferring flavoprotein in Clostridium litoralis and Eubacterium acidaminophilum

Abstract

The glycine-utilizing bacterium Clostridium litoralis contained two enzyme systems for oxidizing dihydrolipoamide. The first one was found to be a genuine dihydrolipoamide dehydrogenase, present only in low amounts. This enzyme had the typical dimeric structure with a subunit molecular mass of about 53 kDa; however, it reacted with both NADP (Km 0.11 mM) and NAD (Km 0.5 mM). The reduction of pyridine nucleotides by dihydrolipoamide was the strongly preferred reaction. A second dihydrolipoamide-oxidizing enzyme system consisted of the interaction of two proteins, the previously described NADP(H)-dependent electron-transferring flavoprotein (D. Dietrichs, M. Meyer, B. Schmidt, and J. R. Andreesen, J. Bacteriol. 172:2088Bacteriol. 172: -2095Bacteriol. 172: , 1990) and a thioredoxin. This enzyme system was responsible for most of the dihydrolipoamide dehydrogenase activity in cell extracts. The thioredoxin did not bind to DEAE, was heat stable, and had a molecular mass of about 15 kDa. N-terminal amino acid analysis of the first 38 amino acid residues resulted in 38% homology to Escherichia coli thioredoxin and about 76% homology to a corresponding protein isolated from the physiologically close related Eubacterium acidaminophilum. The protein of the latter organism had a molecular mass of about 14 kDa and stimulated the low dihydrolipoamide dehydrogenase activity of the corresponding flavoprotein. By this interaction with NADPH-dependent flavoproteins, a new assay system for thioredoxin was established. A function of thioredoxin in glycine metabolism of some anaerobic bacteria is proposed.Eubacterium acidaminophilum and Clostridium litoralis (previously called bacterium W6) are physiologically close related anaerobic organisms specialized with respect to fermentation of glycine and serine if no additional hydrogen donor or acceptor is present. Betaine and sarcosine act only as electron acceptors, thus allowing the utilization of some other amino acids. The two organisms differ with respect to G+C content and some other characteristics (5, 13, 18). Thus, a taxonomic relationship should be excluded, although strong cross-reactivity is observed with use of antibodies raised against proteins involved in the glycine decarboxylase and glycine reductase complexes of E. acidaminophilum (2, 6, 7). In the latter organism, dihydrolipoamide dehydrogenase activity is associated with a protein of exceptionally small molecular size (68 kDa) that is attached to the cytoplasmatic membrane and forms a complex with the selenoprotein PA of glycine reductase (7,8). Therefore, this protein was more thoroughly studied on a comparative basis (3,4,8). The N-terminal sequence data obtained for this protein from E. acidaminophilum and for a corresponding electrontransferring flavoprotein (ET-FP) of Clostridium sporogenes exhibit a strong homology to thioredoxin reductase of Escherichia coli but not to dihydrolipoamide dehydrogenase of other anaerobic bacteria (4). Therefore, we now prefer to call this protein electron-transferring flavopr...

Interaction of selenoprotein PA and the thioredoxin system, components of the NADPH-dependent reduction of glycine in Eubacterium acidaminophilum and Clostridium litorale [corrected]

“…The properties of the latter enzyme, formerly also called electron-transferring flavoprotein (9), suggest that it can be regarded as an atypical thioredoxin reductase (45) or a thioredoxin reductase-like flavoprotein (26) which exhibits some dihydrolipoamide dehydrogenase activity only in E. acidaminophilum (9,26). The demonstration of thioredoxin in E. acidaminophilum and in the physiologically related C. litoralis (26) revives an earlier suggestion of Stadtman (37) of its involvement in the pyridine nucleotide-dependent glycine reductase system. The thioredoxins of E. acidaminophilum and C. litoralis exhibit some pecularities, e.g., lack of binding to DEAE.…”

“…The thioredoxins of E. acidaminophilum and C. litoralis exhibit some pecularities, e.g., lack of binding to DEAE. A new assay for thioredoxin was developed by its interaction with the thioredoxin reductase-like flavoprotein in eliciting new dihydrolipoamide dehydrogenase activity (26). During purification of these proteins from C. litoralis, a protein fraction was obtained which further stimulated the latter activity (26).…”

“…A new assay for thioredoxin was developed by its interaction with the thioredoxin reductase-like flavoprotein in eliciting new dihydrolipoamide dehydrogenase activity (26). During purification of these proteins from C. litoralis, a protein fraction was obtained which further stimulated the latter activity (26). In this report, we describe the identity of that further-activating protein with protein PA of glycine reductase of E. acidaminophilum and C. litoralis as evidenced by biochemical, immunological, and N-terminal sequence data.…”

“…The assay is based on the dihydrolipoamide dehydrogenase activity of the system thioredoxin reductase-like flavoprotein plus thioredoxin that was further stimulated by protein PA (further-stimulation assay). Protein PA was monitored by using an assay that contained, in a final volume of 1 ml; 50 mm potassium phosphate (pH 7.8), 1 mM Na2EDTA, 5 ,ug of thioredoxin reductase-like flavoprotein and 10 ,ug of thioredoxin (purified from E. acidaminophilum and C. litoralis, respectively [16,26]), and 2 mM dihydrolipoamide as an electron donor. After preincubation for 5 to 15 min at 30°C, the reaction was started by adding 0.5 mM NADP+, and the increase of NADPH was monitored at 340 nm.…”

“…Antibodies raised against the thioredoxin reductase-like flavoprotein and thioredoxin were prepared as described previously (16,17). Thioredoxin was prepared as described before (26).…”