Two closely linked genes encoding thioredoxin and thioredoxin reductase in Clostridium litorale.
The genes encoding thioredoxin and thioredoxin reductase of Clostridium litorale were cloned and sequenced. The thioredoxin reductase gene (trxB) encoded a protein of 33.9 kDa, and the deduced amino acid sequence showed 44% identity to the corresponding protein from Escherichia coli. The gene encoding thioredoxin (trxA) was located immediately downstream of trxB. TrxA and TrxB were each encoded by two gene copies, both copies presumably located on the chromosome. Like other thioredoxins from anaerobic, amino-acid-degrading bacteria investigated to date by N-terminal amino acid sequencing, thioredoxin from C. litorale exhibited characteristic deviations from the consensus sequence, e.g., GCVPC instead of WCGPC at the redox-active center. Using heterologous enzyme assays, neither thioredoxin nor thioredoxin reductase were interchangeable with the corresponding proteins of the thioredoxin system from E. coli. To elucidate the molecular basis of that incompatibility, Gly-31 in C. litorale thioredoxin was substituted with Trp (the W in the consensus sequence) by site-directed mutagenesis. The mutant protein was expressed in E. coli and was purified to homogeneity. Enzyme assays using the G31W thioredoxin revealed that Gly-31 was not responsible for the observed incompatibility with the E. coli thioredoxin reductase, but it was essential for activity of the thioredoxin system in C. litorale.
Kreimer
S
S
Institut für Mikrobiologie, Martin-Luther-Universität Halle, Kurt-Mothes-Strasse 3, D-06099 Halle, Germany.
Söhling
B
B
Andreesen
J R
JR
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics* / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase