Characterization of Deinococcus radiophilus thioredoxin reductase active with both NADH and NADPH.
Thioredoxin reductase (TrxR, EC 1.6.4.5) of Deinococcus radiophilus was purified by steps of sonication, ammonium sulfate fractionation, 2'5' ADP Sepharose 4B affinity chromatography, and Sephadex G-100 gel filtration. The purified TrxR, which was active with both NADPH and NADH, gave a 368 U/mg protein of specific activity with 478-fold purification and 18% recovery from the cell-free extract. An isoelectric point of the purified enzymes was ca. 4.5. The molecular weights of the purified TrxR estimated by PAGE and gel filtration were about 63.1 and 72.2 kDa, respectively. The molecular mass of a TrxR subunit is 37 kDa. This suggests that TrxR definitely belongs to low molecular weight TrxR (L-TrxR). The Km and Vmax of TrxR for NADPH are 12.5 μM and 25 μM/min, whereas those for NADH are 30.2 μM and 192 μM/min. The Km and Vmax for 5, 5'-dithio-bis-2-nitrobenzoic acid (DTNB, a substituted substrate for thioredoxin) are 463 μM and 756 μM/min, respectively. The presence of FAD in TrxR was confirmed with the absorbance peaks at 385 and 460 nm. The purified TrxR was quite stable from pH 3 to 9, and was thermo-stable up to 70°C. TrxR activity was drastically reduced (ca. 70%) by Cu(2+), Zn(2+), Hg(2+), and Cd(2+), but moderately reduced (ca. 50%) by Ag(+). A significant inhibition of TrxR by N-ethylmaleimide suggests an occurrence of cysteine at its active sites. Amino acid sequences at the N-terminus of purified TrxR are H(2)N-Ser-Glu-Gln-Ala-Gln-Met-Tyr-Asp-Val-Ile-Ile-Val-Gly-Gly-Gly-Pro-Ala-Gly-Leu-Thr-Ala-COOH. These sequences show high similarity with TrxRs reported in Archaea, such as Methanosarcina mazei, Archaeoglobus fulgidus etc.
Department of Microbiology, Chungbuk National University, Cheongju 361-763, Republic of Korea.
Hee-Jeong
HJ
Seo
Young Nam
YN
Lee
D005591:Chemical Fractionation / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003067:Coenzymes / Q000378:metabolism*; D034301:Deinococcus / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D011485:Protein Binding; D013696:Temperature; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D003067:Coenzymes; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase