On a new artificial mediator accepting NADP(H) oxidoreductase from Clostridium thermoaceticum.
The purification and partial characterisation of an NADP(H) dependent artificial mediator accepting pyridine nucleotide oxidoreductase (AMAPOR) from the anaerobic Clostridium thermoaceticum is described. Depending on the redox potential of the artificial mediators the AMAPOR is able to regenerate NADP+ or NADPH rendering the enzyme useful for preparative work applying NADP(H) dependent oxidoreductases. At 37 degrees C crude extracts of C. thermoaceticum have an AMAPOR activity of 5-7 U mg(-1). This is 28 degrees under the optimal growth temperature of this microrganism. Out of apparently more than 10 AMAPOR active proteins in the crude cell extracts visible after electrophoresis and activity staining on the gel, two of these proteins were isolated. They seem to be two different oligomers. According to gel electrophoresis they show apparent molecular masses of about 200 and 400 kDa. These two forms showed after SDS gel electrophoresis two monomers with apparent molecular masses of 42 and 56 kDa which we call alpha and beta. The two oligomers may have the compositions alpha2beta2 and alpha4beta4. They contain Fe/S cluster and FAD. Various amounts of the FAD were lost during the purification procedure. This loss is partially reversible after addition of FAD. The AMAPOR reacts with rather different artificial mediators such as viologens, quinones e.g. 1,4-benzoquinone or anthraquinone-2,6-disulphonate, 2,6-dichloro-indophenol and clostridial rubredoxin. Two different ferredoxins from C. thermoaceticum, oxygen or lipoamide are no substrates indicating the here described AMAPOR is not a diaphorase in the usual sense.
Günther
H
H
Institut für Organische Chemie und Bioehemie, Technische Universität München, Garching, Germany.
Walter
K
K
Köhler
P
P
Simon
H
H
D000595:Amino Acid Sequence; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology* / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005182:Flavin-Adenine Dinucleotide / Q000302:isolation & purification / Q000378:metabolism; D005415:Flavins / Q000032:analysis; D007501:Iron / Q000032:analysis; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D013455:Sulfur / Q000032:analysis
D005415:Flavins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D013455:Sulfur; D007501:Iron; D009247:NADH, NADPH Oxidoreductases; C418411:artificial mediator accepting pyridine nucleotide oxidoreductase