Characterization of a thioredoxin-thioredoxin reductase system from the hyperthermophilic bacterium Thermotoga maritima.
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.
Yang
Xianqin
X
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada. kma@uwaterloo.ca.
Ma
Kesen
K
D000818:Animals; D001594:Benzyl Viologen / Q000378:metabolism; D002417:Cattle; D003067:Coenzymes / Q000032:analysis / Q000378:metabolism; D019281:Dimerization; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013879:Thioredoxins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D003067:Coenzymes; D007328:Insulin; D009243:NAD; D001594:Benzyl Viologen; D005182:Flavin-Adenine Dinucleotide; D013879:Thioredoxins; D009249:NADP; D004228:Dithionitrobenzoic Acid; D013880:Thioredoxin-Disulfide Reductase