Purification and partial characterisation of a reversible artificial mediator accepting NADH oxidoreductase from Clostridium thermoaceticum.
An NAD(H)-dependent artificial mediator accepting pyridine nucleotide oxidoreductase present in Clostridium thermoaceticum has been purified 50-fold by three chromatographic steps to apparent electrophoretical homogeneity with a yield of 25%. By PAGE and gel filtration the molecular mass of the native enzyme was estimated to be 200 kDa and 210 kDa, respectively. By SDS/gel electrophoresis, a single band was found at 17000 Da, suggesting a homododecamer. Reducing carbamoylmethylviologen or hexacyanoferrate(III) with NADH, the enzyme was most active at pH 10 and the specific activities were 100 mumol min-1 mg-1 protein and 800 mumol min-1 mg-1 protein, respectively. The K(m) values for hexacyanoferrate(III), carbamoylmethylviologen and NADH at pH 8.5 were determined to be 0.40, 0.55 and 1.1 mM, respectively. Other electron acceptors for the dehydrogenation of NADH were 2,6-dichlorophenolindophenol, anthraquinone-2,6-disulphonate, ubiquinone 0 and FAD. In the reduction of NAD+ with reduced methyl viologen (MV+), the specific activity was about 225 mumol min-1 mg-1 protein at the pH maximum of 5.0. The K(m) values for reduced methylviologen, NADH and NAD+ were 1.0, 1.1 and 0.25 mM, respectively. The enzyme had 10.6 atoms iron and 12.7 atoms sulphur per dodecamer. A significant content of flavin or molybdopterin cofactor could not be detected. The first 45 amino acids of the oxidoreductase show a surprisingly high degree of identity or similarity with the ribosomal L12 protein of various eubacteria, the acyl carrier proteins of microorganisms, but also with bovine heart mitochondria and a 3-phosphoglycerate dehydrogenase as well as a gyceraldehyde-3-phosphate dehydrogenase from bacteria and pea chloroplasts, respectively.
Bayer
M
M
Institut für Organische Chemie und Biochemie, Technische Universität München, Germany.
Walter
K
K
Simon
H
H
D015086:2,6-Dichloroindophenol / Q000378:metabolism; D000595:Amino Acid Sequence; D000880:Anthraquinones / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology*; D004795:Enzyme Stability; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007501:Iron / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000302:isolation & purification*; D010269:Paraquat / Q000031:analogs & derivatives; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur / Q000032:analysis
D000880:Anthraquinones; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D013455:Sulfur; D005486:Flavin Mononucleotide; D015086:2,6-Dichloroindophenol; D007501:Iron; D009250:NADP Transhydrogenases; D010269:Paraquat