Fast purification of thioredoxin reductases and of thioredoxins with an unusual redox-active centre from anaerobic, amino-acid-utilizing bacteria.
Thioredoxin reductase and thioredoxin are primarily involved in catabolic metabolism as important electron carriers in anaerobic, amino-acid-degrading bacteria. A general and fast procedure was developed for the purification of thioredoxin reductase and thioredoxin from Eubacterium acidaminophilum, Clostridium litorale, C. sticklandii, C. sporogenes, C. cylindrosporum and 'Tissierella creatinophila' based upon their properties: the binding to 2',5'-AMP-Sepharose by thioredoxin reductase and the inability of thioredoxins to bind to a DEAE-Sephacel column. The consensus sequence at the active site of thioredoxins (-WCGPC-) was found to be modified in all of these anaerobes: Trp-31 (Escherichia coli nomenclature) was replaced by Gly or Ser, Gly-33 by Val or Glu. None of these thioredoxins reacted with thioredoxin reductase of E. coli or vice versa, but they did interact with the thioredoxin reductases obtained from the other anaerobes studied. Based upon their distinguishing features it is suggested that these thioredoxins might form an evolutionarily separate group.
Harms
Claudia
C
Institut für Mikrobiologie der Georg-August-Universität Göttingen, Grisebachstr. 8, 37077 Göttingen, Germany.
Meyer
Manfred A
MA
Institut für Mikrobiologie der Georg-August-Universität Göttingen, Grisebachstr. 8, 37077 Göttingen, Germany.
Andreesen
Jan R
JR
Institut für Mikrobiologie der Martin-Luther-Universität Halle, Kurt-Mothes-Str. 3, 06099 Halle, >Germany.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D002847:Chromatography, Agarose; D003013:Clostridium / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D016384:Consensus Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005051:Eubacterium / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D006089:Gram-Negative Anaerobic Bacteria / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017421:Sequence Analysis; D013880:Thioredoxin-Disulfide Reductase / Q000302:isolation & purification* / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D007328:Insulin; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase