Cloning, expression, and nucleotide sequence of a gene encoding a second thioredoxin from Corynebacterium nephridii.

A gene encoding thioredoxin in Corynebacterium nephridii was cloned in Escherichia coli by complementation of a thioredoxin mutant. Transformants that appeared to complement were analyzed for the presence of thioredoxin by the coupled assay using methionine sulfoxide reductase. Of 18 transformants, four contained high levels of thioredoxin activity. Transformants containing plasmids pLCN2 and pLCN4 were unable to support replication of T7 phage, in spite of their thioredoxin activities, and were studied in more detail. The plasmid pLCN2 contains a 1.85-kilobase Sau3AI insert, whereas pLCN4 contains a 10-kilobase TaqI insert. These plasmids complement all phenotypes of a thioredoxin-deficient strain except for replication of T7 phage. The nucleotide sequence of a 620-base pair HinfI fragment encoding thioredoxin derived from either plasmid indicated that the protein derived from this DNA is different from the thioredoxin of C. nephridii previously reported (Meng, M., and Hogenkamp, H.P.C. (1981) J. Biol. Chem. 256, 9174-9182). The amino acid sequence predicted from the nucleotide sequence shows a high degree of homology with other procaryotic thioredoxins. However, the new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site and a third half-cystine residue in the carboxyl-terminal domain of the protein. The molecular weight of this thioredoxin, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is smaller than that estimated from the DNA sequence, suggesting that processing may have occurred.