Characterization of a thioredoxin-thioredoxin reductase system from the hyperthermophilic bacterium Thermotoga maritima

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 micromol NADH oxidized min(-1)mg(-1) and 115 +/- 2.4 micromol NADPH oxidized min(-1)mg(-1), respectively. The apparent K(m) values were determined to be 89 +/- 1.1 microM, 73 +/- 1.6 microM, and 780 +/- 20 microM 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 degrees C, and more than 60% of the activity remained after incubation for 28 h at 80 degrees 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.

FIG. 1.

SDS-PAGE (12.5%) of the purified thioredoxin reductase (TR) and thioredoxin (Trx) from Thermotoga maritima. Lanes 1 and 5, low-molecular-mass standards along with their corresponding molecular masses; lane 2, purified T. maritima TR (1 μg); lanes 3 and 4, purified T. maritima Trx (0.4 and 0.6 μg, respectively).

FIG. 2.

pH dependency of the purified thioredoxin reductase (TR) from Thermotoga maritima. The activity was assayed at 80°C with NADH (open symbols) or NADPH (filled symbols) as an electron donor. Circles, 100 mM sodium phosphate buffer, pH 6.0 to 8.0; triangles, 100 mM glycylglycine-NaOH buffer, pH 8.0 to 9.0; diamonds, 100 mM glycine-NaOH buffer, pH 8.5 to 10.0; squares, 100 mM CAPS buffer, pH 10.0 to 11.0; inverted triangles, 100 mM sodium phosphate buffer, pH 11.0 to 12.0.

FIG. 3.

Reduction of insulin by Thermotoga maritima thioredoxin (Trx). The assay mixture contained 1 mM dithiothreitol and 1 mg/ml insulin in 100 mM sodium phosphate buffer, pH 7.0. The reaction was carried out at 30°C by monitoring the increase of absorbance at 650 nm in the absence (open circles) or presence of 0.15 μM (filled circles), 0.29 μM (filled triangles), or 0.58 μM (open triangles) T. maritima Trx.

FIG. 4.

Reduction of insulin by the Thermotoga maritima thioredoxin system. The assay mixture contained 50 nM T. maritima thioredoxin reductase, 0.2 mM NADH (A) or NADPH (B), 0.13 mM insulin, 1 mM EDTA, and 0 μM (open circles), 0.36 μM (crosses), 0.72 μM (diamonds), or 1.44 μM (triangles) T. maritima thioredoxin in 100 mM sodium phosphate buffer, pH 7.0. The increase of absorbance at 650 nm was monitored at 30°C.

FIG. 5.

Reduction of DTNB by the Thermotoga maritima thioredoxin system. The assay mixture contained 50 nM T. maritima thioredoxin reductase, 0.2 mM NADH (open circles) or NADPH (filled circles), 0.1 mM DTNB, and 0 to 1.4 μM T. maritima thioreodxin. The increase of absorbance at 412 nm was monitored at 30°C.