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(Received for publication, July 23, 1997, and in revised form, September 26, 1997)
From the Department of Biosciences at Novum, Karolinska Institute,
S-141 57 Huddinge, Sweden
Thioredoxin (Trx) is a small ubiquitous protein that
displays different functions mainly via redox-mediated processes. We here report the cloning of a gene (trxC) coding for a novel
thioredoxin in Escherichia coli as well as the expression
and characterization of its product. The gene encodes a protein of 139 amino acids (Trx2) with a calculated molecular mass of 15.5 kDa. Trx2
contains two distinct domains: an N-terminal domain of 32 amino acids
including two CXXC motifs and a C-terminal domain, with the
conserved active site, Trp-Cys-Gly-Pro-Cys, showing high homology to
the prokaryotic thioredoxins. Trx2 together with thioredoxin reductase
and NADPH is an efficient electron donor for the essential enzyme
ribonucleotide reductase and is also able to reduce the interchain
disulfide bridges of insulin. The apparent Km value
of Trx2 for thioredoxin reductase is similar to that of the previously
characterized E. coli thioredoxin (Trx1). The enzymatic
activity of Trx2 as a protein-disulfide reductase is increased by
preincubation with dithiothreitol, suggesting that oxidation of
cysteine residues other than the ones in the active site might regulate
its activity. A truncated form of the protein, lacking the N-terminal
domain, is insensitive to the presence of dithiothreitol, further
confirming the involvement of the additional cysteine residues in
modulating Trx2 activity. In addition, the presence of the N-terminal
domain appears to confer heat sensitivity to Trx2, unlike Trx1.
Finally, Trx2 is present normally in growing E. coli cells
as shown by Western blot analysis.
Volume 272, Number 49,
Issue of December 5, 1997
pp. 30841-30847
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning, Expression, and Characterization of a Novel
Escherichia coli Thioredoxin
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