Three different dihydrolipoamide dehydrogenases were purified to homogenity from the anaerobic glycine-utilizing bacteria Clostridium cylindrosporum, Clostridium sporogenes, and Peptostreptococcus glycinophilus, and their basic properties were determined. The enzyme isolated from P. glycinophilus showed the properties typical of dihydrolipoamide dehydrogenases: it was a dimer with a subunit molecular mass of 53,000 and contained 1 mol of flavin adenine dinucleotide and 2 redox-active sulfhydryl groups per subunit. Only NADH was active as a coenzyme for reduction of lipoamide. Spectra of the oxidized enzyme exhibited maxima at 230, 270, 353, and 453 nm, with shoulders at 370, 425, and 485 nm. The dihydrolipoamide dehydrogenases of C. cylindrosporum and C. sporogenes were very similar in their structural properties to the enzyme of P. glycinophilus except for their coenzyme specificity. The enzyme of C. cylindrosporum used NAD(H) as well as NADP(H), whereas the enzyme of C. sporogenes reacted only with NADP(H), and no reaction could be detected with NAD(H). Antibodies raised against the dihydrolipoamide dehydrogenase of C. cylindrosporum reacted with extracts of Clostridium acidiurici, Clostridium purinolyticum, and Eubacterium angustum, whereas antibodies raised against the enzymes of P. glycinophilus and C. sporogenes showed no cross-reaction with extracts from 42 organisms tested.

Purification of protein PA of the glycine reductase complex from Eubacterium acidaminophUum and Clostridium litoralis was monitored by a new spectrophotometric assay. The procedure depended on a specific two-to threefold stimulation of a dihydrolipoamide dehydrogenase activity that is elicited by the interaction of a thioredoxin reductase-like flavoprotein and thioredoxin from both organisms. Protein PA isolated from E.acidaminophilum by 75 Se iabeling and monitoring of the dithioerythritol-dependent glycine reductase activity was identical in its biochemical, structural, and immunological properties to the protein isolated by using the stimulation assay. Proteins PA from both organisms were glycoproteins of Mr about 18,500 and exhibited very similar N-terminal amino acid sequences. Depletion of thioredoxin from crude extracts of E. acidaminophilum totally diminished the NADPH-dependent but not the dithioerythritol-dependent glycine reduction. The former activity could be fully restored by adding thioredoxin. Antibodies raised against the thioredoxin reductase-like flavoprotein or thioredoxin inhibited to a high extent NADPH-dependent but not dithioerythritol-dependent glycine reductase activity. These results indicate the involvement of the thioredoxin system in the electron flow from reduced pyridine nucleotides to glycine reductase.

The lipoamide dehydrogenase of the glycine decarboxylase complex was purified to homogeneity (8 U/mg) from cells of the anaerobe Eubacterium acidaminophilum that were grown on glycine. In cell extracts four radioactive protein fractions labeled with D-[2-'4C]riboflayin could be detected after gel filtration, one of which coeluted with lipoamide dehydrogenase activity. The of its procaryotic or eucaryotic origin (4,5,14,18,30,33,50,53,55). The multiplicity of isoenzymes observed from eucaryotic origin was found to be a conformational isomerism (19). Immunological studies suggest that lipoamide dehydrogenase is identical in the pyruvate, 2-oxoglutarate, and branched-chain 2-oxoacid dehydrogenases of the rat heart (26).In enzyme assays pig heart diaphorase is able to replace lipoamide dehydrogenase, protein L, from the chicken liver glycine decarboxylase (12). The lipoamide dehydrogenase of glycine decarboxylase from pea leaf mitochondria exhibits a monomer with an Mr of about 59,000, which is similar to that of the enzyme that is involved in the pyruvate dehydrogenase complex, and monoclonal antibodies raised against lipoamide dehydrogenase inhibit both enzymes to the same extent (51). Therefore, being a conservative enzyme, lipoamide dehydrogenase has also been used to calculate evolutionary relationships (9, 27) to other pyridine nucleotide disulfide oxidoreductase flavoproteins such as glutathione reductase, thioredoxin reductase, and mercuric reductase (11,16,36,54).As the only exception Pseudomonas putida and Pseudomonas aeruginosa produce two lipoamide dehydrogenases during growth on valine, which differ to some degree in molecular masses and kinetic parameters. One is part of 2-oxoglutarate dehydrogenase and probably pyruvate dehydrogenase; the other is part of branched-chain 2-oxoacid dehydrogenase (27,39). During glycine oxidation by P. putida, only the lipoamide dehydrogenase of 2-oxoglutarate dehydrogenase is involved, which has a monomer molecular mass of 56 kilodaltons (kDa), however, rather than the enzyme specific for valine, which has a monomer molecular mass of 49 kDa (38).From all these data it seemed that no specific lipoamide dehydrogenase is involved in glycine oxidation. However, during the isolation of the glycine decarboxylase proteins

Three electron-transferring flavoproteins were purified to homogeneity from anaerobic, amino acid-utilizing bacteria (bacterium W6, Clostridium sporogenes, and Clostridium sticklandii), characterized, and compared with the dihydrolipoamide dehydrogenase of Eubacterium acidaminophilum. All the proteins were found to be dimers consisting of two identical subunits with a subunit Mr of about 35,000 and to contain about 1 mol of flavin adenine dinucleotide per subunit. Spect.ra of the oxidized proteins exhibited characteristic absorption of flavoproteins, and the reduced proteins showed an A580 indicating a neutral'semiquinone. Many artificial electron acceptors, including methyl viologen, could be used with NADPH as the electron donor but not with NADH. Unlike the enzyme'of E. acidaminophilum, which exhibited by itself a dihydrolipoamide dehydrogenase activity (W. Freudenberg, D. Dietrichs, H. Lebertz, and J. R. Andreesen, J. Bacteriol. 171:1346Bacteriol. 171: -1354Bacteriol. 171: , 1989, the electron-transferring flavoprotein purified from bacterium W6 reacted with lipoamide only under certain assay conditions, whereas the proteins of C. sporogenes and C. sticklandii exhibited no dihydrolipoamide dehydrogenase activity. The three homogeneous electron-transferring flavoproteins were very similar in their structural and biochemical properties to the dihydrolipoamide dehydrogenase of E. acidaminophilum and exhibited cross-reaction with antibodies raised against the latter enzyme. N-terminal sequence analysis demonstrated a high degree of homology between the dihydrolipoamide dehydrogenase of E. acidaminophilum and the electron-transferring fiavoprotein of C. sporogenes to the thioredoxin reductase of Escherichia coli. Unlike these proteins, the dihydrolipoamide dehydrogenases purified from' the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and C. sporogenes exhibited a high homology to dihydrolipoamide dehydrogenases known from other organisms.

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...