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- Characterization of Deinococcus radiophilus Thioredoxin Reductase Active with Both NADH and NADPH
- Hee-Jeong Seo , Young Nam Lee
- J. Microbiol. 2010;48(5):637-643. Published online November 3, 2010
- DOI: https://doi.org/10.1007/s12275-010-0283-7
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Abstract
- 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 μ M/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 Cu2+, Zn2+, Hg2+, and Cd2+, 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 H2N-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.
- Isoforms of Glucose 6-Phosphate Dehydrogenase in Deinococcus radiophilus
- Ji Youn Sung , Young Nam Lee
- J. Microbiol. 2007;45(4):318-325.
- DOI: https://doi.org/2566 [pii]
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Abstract
- Glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in Deinococcus radiophilus, an extraordinarily UV-resistant bacterium, was investigated to gain insight into its resistance as it was shown to be involved in a scavenging system of superoxide (O2-1) and peroxide (O2-2) generated by UV and oxidative stresses. D. radiophilus possesses two G6PDH isoforms: G6PDH-1 and G6PDH-2, both showing dual coenzyme specificity for NAD and NADP. Both enzymes were detected throughout the growth phase; however, the substantial increase in G6PDH-1 observed at stationary phase or as the results of external oxidative stress indicates that this enzyme is inducible under stressful environmental conditions. The G6PDH-1 and G6PDH-2 were purified 122- and 44-fold (using NADP as cofactor), respectively. The purified G6PDH-1 and G6PDH-2 had the specific activity of 2,890 and 1,033 U/mg protein (using NADP as cofactor) and 3,078 and 1,076 U/mg protein (using NAD as cofactor), respectively. The isoforms also evidenced distinct structures; G6PDH-1 was a tetramer of 35 kDa subunits, whereas G6PDH-2 was a dimer of 60 kDa subunits. The pIs of G6PDH-1 and G6PDH-2 were 6.4 and 5.7, respectively. Both G6PDH-1 and G6PDH-2 were inhibited by both ATP and oleic acid, but G6PDH-1 was found to be more susceptible to oleic acid than G6PDH-2. The profound inhibition of both enzymes by β-naphthoquinone-4-sulfonic acid suggests the involvement of lysine at their active sites. Cu2+ was a potent inhibitor to G6PDH-2, but a lesser degree to G6PDH-1. Both G6PDH-1 and G6PDH-2 showed an optimum activity at pH 8.0 and 30°C.
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