Affiliation:
1. Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada
Abstract
ABSTRACT
A thioredoxin reductase and a thioredoxin were purified to homogeneity from a cell extract of
Thermotoga maritima
. The thioredoxin reductase was a homodimeric flavin adenine dinucleotide (FAD)-containing protein with a subunit of 37 kDa estimated using SDS-PAGE, which was identified to be TM0869. The amino acid sequence of the enzyme showed high identities and similarities to those of typical bacterial thioredoxin reductases. Although the purified
T. maritima
thioredoxin reductase could not use thioredoxin from
Spirulina
as an electron acceptor, it used thioredoxin that was purified from
T. maritima
by monitoring the dithiothreitol-dependent reduction of bovine insulin. This enzyme also catalyzed the reduction of benzyl viologen using NADH or NADPH as an electron donor with apparent
V
max
values of 1,111 ± 35 μmol NADH oxidized min
−1
mg
−1
and 115 ± 2.4 μmol NADPH oxidized min
−1
mg
−1
, respectively. The apparent
K
m
values were determined to be 89 ± 1.1 μM, 73 ± 1.6 μM, and 780 ± 20 μM for benzyl viologen, NADH, and NADPH, respectively. Optimal pH values were determined to be 9.5 and 6.5 for NADH and NADPH, respectively. The enzyme activity increased along with the rise of temperature up to 95°C, and more than 60% of the activity remained after incubation for 28 h at 80°C. The purified
T. maritima
thioredoxin was a monomer with a molecular mass of 31 kDa estimated using SDS-PAGE and identified as TM0868, which exhibited both thioredoxin and thioltransferase activities.
T. maritima
thioredoxin and thioredoxin reductase together were able to reduce insulin or 5,5′-dithio-bis(2-nitrobenzoic acid) using NAD(P)H as an electron donor. This is the first thioredoxin-thioredoxin reductase system characterized from hyperthermophilic bacteria.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
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