CLONING, EXPRESSION, AND CHARACTERIZATION OF A NOVEL ESCHERICHIA-COLITHIOREDOXIN

Thioredoxin (Trx) is a small ubiquitous protein that displays differen t functions mainly via redox mediated processes, We here report the cl oning of a gene (trxC) coding for a novel thioredoxin in Escherichia c oli as well as the expression and characterization of its product. The gene encodes a protein of 139 amino acids (Trx2) with a calculated mo lecular mass of 15.5 kDa, Trx2 contains two distinct domains: an N-ter minal domain of 32 amino acids including two CXXC motifs and a C-termi nal domain, with the conserved active site, Trp-Cys-Gly-Pro-Cys, showi ng high homology to the prokaryotic thioredoxins, Trx2 together with t hioredoxin reductase and NADPH is an efficient electron donor for the essential enzyme ribonucleotide reductase and is also able to reduce t he interchain disulfide bridges of insulin. The apparent K-m value of Trx2 for thioredoxin reductase is similar to that of the previously ch aracterized E. coli thioredoxin (Trx1). The enzymatic activity of Trx2 as a protein-disulfide reductase is increased by preincubation with d ithiothreitol, suggesting that oxidation of cysteine residues other th an the ones in the active site might regulate its activity. A truncate d form of the protein, lacking the N-terminal domain, is insensitive t o the presence of dithiothreitol, further confirming the involvement o f the additional cysteine residues in modulating Trx2 activity. In add ition, the presence of the N-terminal domain appears to confer heat se nsitivity to Trx2, unlike Trx1. Finally, Trx2 is present normally in g rowing E. coli cells as shown by Western blot analysis.