Purification of NADPH-dependent electron-transferring flavoproteins and N-terminal protein sequence data of dihydrolipoamide dehydrogenases from anaerobic, glycine-utilizing bacteria

Abstract

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.

Recombinant expression, characterization and application of a dihydrolipoamide dehydrogenase with diaphorase activity from Bacillus sphaericus

“…As seen in Table 1, none of them enhanced or inhibited the enzyme activity. This was similar to the results achieved by other diaphorases (Dietrichs et al 1990;Argyrou et al 2003). Kinetic constants of the recombinant purified enzyme for NADH substrate were determined.…”

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 involvement of a thioredoxin reductase-like flavoprotein in the pyridine nucleotide-dependent reduction of glycine could be assumed after it was shown by double immunocytochemical labeling studies that there was a close association of that protein with protein PA of glycine reductase in E. acidaminophilum (9,17). The existence of a thioredoxin was successively demonstrated for C. litoralis and E. acidaminophilum (26).…”

Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system

“…The N-terminal amino acid sequence of DHLDH from P. carbinolicus exhibited a high degree of homology to DHLDHs of different origin (Table 2), including the FADbinding site (position 13 to 18), the sequence of which is known from other pyridine nucleotide-disulfide oxidoreductases (7,12).…”