Glutamyl-tRNA reductase of Chlorobium vibrioforme is a dissociable homodimer that contains one tightly bound heme per subunit
Glutamyl-tRNA reductase of Chlorobium vibrioforme is a dissociable homodimer that contains one tightly bound heme per subunit
复制标题
DOI：
10.1128/jb.187.13.4444-4450.2005
复制
发表时间：
2005-07-01
影响因子：
3.2
通讯作者：
Beale, SI
中科院分区：
文献类型：
--
作者：
Srivastava, A;Beale, SI
在绿色硫磺细菌Chlorobium vibrioforme中，以谷氨酸为原料，通过依赖tRNA的五碳途径合成了四吡咯的生物合成前体--D-氨基乙酰丙酸。由HEMA基因编码的谷氨酰-tRNA还原酶(GTR)催化了该途径的第一步，即tRNA结合的谷氨酸还原为谷氨酸i-半醛。将弧菌的HEMA基因克隆到表达载体中，并在表达的蛋白上添加一个N-末端的组氨酸标签，从而对GTR蛋白进行鉴定。用镍亲和柱层析纯化His标记的GTR蛋白。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳板(SDS-PAGE)上可见一条约49 kDa的条带。凝胶过滤层析测定其天然相对分子质量约为40 kDa，表明天然GTR为单体。然而，当该蛋白与5%(VOL/VOL)甘油混合时，产物的表观分子质量为95 kDa，表明在此条件下该蛋白为二聚体。纯化的His(6)-GTR与大肠杆菌谷氨酰tRNA(Glu)和纯化的重组莱茵衣藻谷氨酸-1-半醛转氨酶(Chlamydomonas rehardtii Glamate-1-半醛氨基转移酶)温育后，体外具有催化活性。每摩尔肽亚基含有1摩尔紧密结合的血红素。在整个纯化过程中，血红素仍与蛋白质结合，不会被阴离子或阳离子交换柱层析除去。然而，如果蛋白质在β-巯基乙醇存在下变性，结合的血红素在SDS-PAGE中被释放。在体外，添加亚铁血红素不能抑制纯化表达的GTR的活性。然而，当在血红素合成抑制剂3-氨基-2，3-二氢苯甲酸(Gabaculine)存在下表达GtR时，纯化的gtr结合的血红素比对照减少了60%~70%，并且在体外被氯化血红素抑制。
delta-Aminolevulinic acid, the biosynthetic precursor of tetrapyrroles, is synthesized from glutamate via the tRNA-dependent five-carbon pathway in the green sulfur bacterium Chlorobium vibrioforme. The enzyme glutamyl-tRNA reductase (GTR), encoded by the hemA gene, catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate I-semialdehyde. To characterize the GTR protein, the hemA gene from C vibrioforme Was cloned into expression plasmids that added an N-terminal His, tag to the expressed protein. The His-tagged GTR protein was purified using Ni affinity column chromatography. GTR was observable as a 49-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The native molecular mass, as determined by gel filtration chromatography, appeared to be approximately 40 kDa, indicating that native GTR is a monomer. However, when the protein was mixed with 5 % (vol/vol) glycerol, the product had an apparent molecular mass of 95 kDa, indicating that the protein is a dimer under these conditions. Purified His(6)-GTR was catalytically active in vitro when it was incubated with Escherichia coli glutamyl-tRNA(Glu) and purified recombinant Chlamydomonas reinhardtii glutamate-1-semialdehyde aminotransferase. The expressed GTR contained 1 mol of tightly bound heme per mol of peptide subunit. The heme remained bound to the protein throughout purification and was not removed by anion- or cation-exchange column chromatography. However, the bound heme was released during SDS-PAGE if the protein was denatured in the presence of beta-mercaptoethanol. Added heme did not inhibit the activity of purified expressed GTR in vitro. However, when the GTR was expressed in the presence of 3-amino-2,3-dihydrobenzoic acid (gabaculine), an inhibitor of heme synthesis, the purified GTR had 60 to 70 % less bound heme than control GTR, and it was inhibited by hemin in vitro.