Purification and comparative studies of dihydrolipoamide dehydrogenases from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and Clostridium sporogenes

Abstract

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.

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

“…E. acidaminophilum was anaerobically grown as described before (23,46). Acetobacteroides glycinophilus, C. litoralis (bacterium W6), C. cylindrosporum, C. sporogenes, C. sticklandii, P. glycinophilus, P. parvulus, P. variabilis, and Peptostreptococcus strain NCTC 9811 of the anaerobic Hare group IX were grown as described previously (8). C. cochlearium was grown anaerobically in a complex medium as described before (21), and Sporomusa ovata was grown in a medium containing 50 mM betaine (27).…”

“…Preparation of cell extracts. Cell extracts of different organisms were prepared as described before (8).…”

Evidence for the functional importance of Cys298 in D‐amino acid oxidase from Trigonopsis variabilis

“…The first injection contained 300 pg protein and the booster injection was carried out with 230 pg enzyme. Antibodies were purified as described before [33]. Western immunoblots were performed in analogy to ~5 1 .…”

Cloning, Sequencing and Functional Expression of Dihydrolipoamide Dehydrogenase from the Human Pathogen Trypanosoma Cruzi

“…As part of these complexes, LipDH catalyzes the reoxidation of protein-bound dihydrolipoyl residues: dihydrolipoamide + NAD' -lipoamide + NADH + Hi LipDH is ubiquitous in aerobic organisms. However, its occurence in anaerobic organisms (Dietrichs and Andreesen, 1990) and in bloodstream African trypanosomes, which lack a functional mitochondrion (Danson et al, 1987), led to the proposal that the function of LipDH is not restricted to its roles within the multienzyme complexes. From the insect parasite Crithidia fasciculata the enzyme has recently been characterized as part of the pyruvate dehydrogenase complex (Diaz and Komuniecki, 1995).…”