Department of Chemistry, University of Saskatchewan, Saskatoon S7N 5C9, Canada.
J Bacteriol. 2010 Jan;192(2):494-501. doi: 10.1128/JB.01046-09. Epub 2009 Nov 20.
This paper describes the cloning, purification, and characterization of thioredoxin (Trx) and thioredoxin reductase (TrxR) and the structure determination of TrxR from the ionizing radiation-tolerant bacterium Deinococcus radiodurans strain R1. The genes from D. radiodurans encoding Trx and TrxR were amplified by PCR, inserted into a pET expression vector, and overexpressed in Escherichia coli. The overexpressed proteins were purified by metal affinity chromatography, and their activity was demonstrated using well-established assays of insulin precipitation (for Trx), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reduction, and insulin reduction (for TrxR). In addition, the crystal structure of oxidized TrxR was determined at 1.9-A resolution. The overall structure was found to be very similar to that of E. coli TrxR and homodimeric with both NADPH- and flavin adenine dinucleotide (FAD)-binding domains containing variants of the canonical nucleotide binding fold, the Rossmann fold. The K(m) (5.7 muM) of D. radiodurans TrxR for D. radiodurans Trx was determined and is about twofold higher than that of the E. coli thioredoxin system. However, D. radiodurans TrxR has a much lower affinity for E. coli Trx (K(m), 44.4 muM). Subtle differences in the surface charge and shape of the Trx binding site on TrxR may account for the differences in recognition. Because it has been suggested that TrxR from D. radiodurans may have dual cofactor specificity (can utilize both NADH and NADPH), D. radiodurans TrxR was tested for its ability to utilize NADH as well. Our results show that D. radiodurans TrxR can utilize only NADPH for activity.
本文描述了耐辐射球菌（Deinococcus radiodurans）菌株 R1 中的硫氧还蛋白（Trx）和硫氧还蛋白还原酶（TrxR）的克隆、纯化和特性，以及 TrxR 的结构测定。通过 PCR 扩增来自耐辐射球菌的编码 Trx 和 TrxR 的基因，将其插入 pET 表达载体中，并在大肠杆菌中过表达。通过金属亲和层析纯化过表达的蛋白质，并使用胰岛素沉淀（用于 Trx）、5,5'-二硫代双（2-硝基苯甲酸）（DTNB）还原和胰岛素还原（用于 TrxR）的成熟测定法证明其活性。此外，还确定了氧化型 TrxR 的晶体结构，分辨率为 1.9A。整体结构与大肠杆菌 TrxR 非常相似，为同源二聚体，均包含 NADPH 和黄素腺嘌呤二核苷酸（FAD）结合域，具有典型核苷酸结合折叠的变体，即 Rossmann 折叠。测定了 D. radiodurans TrxR 对 D. radiodurans Trx 的 K(m)（5.7 μM），约为大肠杆菌硫氧还蛋白系统的两倍。然而，D. radiodurans TrxR 对大肠杆菌 Trx 的亲和力要低得多（K(m)，44.4 μM）。TrxR 上 Trx 结合位点的表面电荷和形状的细微差异可能导致识别的差异。由于有人提出耐辐射球菌的 TrxR 可能具有双重辅因子特异性（可以利用 NADH 和 NADPH），因此还测试了 D. radiodurans TrxR 利用 NADH 的能力。我们的结果表明，D. radiodurans TrxR 只能利用 NADPH 发挥活性。
