2 CLOSELY LINKED GENES ENCODING THIOREDOXIN AND THIOREDOXIN REDUCTASEIN CLOSTRIDIUM-LITORALE

The genes encoding thioredoxin and thioredoxin reductase of Clostridiu m litorale were cloned and sequenced. The thioredoxin reductase gene ( trxB) encoded a protein of 33.9 kDa, and the deduced amino acid sequen ce showed 44% identity to the corresponding protein from Escherichia c oli. The gene encoding thioredoxin (trxA) was located immediately down stream of trxB. TrxA and TrxB were each encoded by two gene copies, bo th copies presumably located on the chromosome. Like other thioredoxin s from anaerobic, amino-acid-degrading bacteria investigated to date b y N-terminal amino acid sequencing, thioredoxin from C. litorale exhib ited characteristic deviations from the consensus sequence, e.g., GCVP C instead of WCGPC at the redox-active center. Using heterologous enzy me assays, neither thioredoxin nor thioredoxin reductase were intercha ngeable with the corresponding proteins of the thioredoxin system from E. coli. To elucidate the molecular basis of that incompatibility, Gl y-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 assay s using the G31W thioredoxin revealed that Gly-31 was not responsible for the observed incompatibility with the E. coli thioredoxin reductas e, but it was essential for activity of the thioredoxin system in C. l itorale.