Self W T, Hasona A, Shanmugam K T
Department of Microbiology and Cell Science, Box 110700, University of Florida, Gainesville, FL 32611, USA.
Microbiology (Reading). 2001 Nov;147(Pt 11):3093-104. doi: 10.1099/00221287-147-11-3093.
The formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H2 and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers. The structural genes of this enzyme complex are activated by the FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhlA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the hyc operon. Analysis of the N-terminal sequence of the FhlA protein revealed a unique motif (amino acids 7-37), which is also found in ATPases associated with several members of the ABC-type transporter family. A deletion derivative of FhlA lacking these amino acids (FhlA9-2) failed to activate the hyc operon in vivo, although the FhlA9-2 did bind to hyc promoter DNA in vitro. The ATPase activity of the FhlA9-2-DNA-formate complex was at least three times higher than that of the native protein-DNA-formate complex, and this degree of activity was achieved at a lower formate level. Extending the deletion to amino acid 117 (FhlA167) not only reversed the FhlA(-) phenotype of FhlA9-2, but also led to both molybdenum- and formate-independence. Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhlA165), which had a twofold higher level of hyc operon expression than the native protein. Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of hyc-lac expression. The FhlA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhlA protein, while the FhlA167 protein had a significantly lower affinity for hyc promoter DNA in vitro. Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhlA165 or FhlA167 responded to formate. Removal of the first 117 amino acids of the FhlA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex. The sequence similarity to ABC-ATPases, combined with the properties of the FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.
大肠杆菌的甲酸氢裂解酶复合物催化甲酸裂解为二氧化碳和氢气，它由钼酶甲酸脱氢酶-H、氢化酶3和中间电子载体组成。该酶复合物的结构基因在同时存在甲酸和钼酸盐的情况下被FhlA蛋白激活；ModE-Mo作为二级激活剂。对FhlA蛋白的突变分析表明，该蛋白独特的N端区域负责hyc操纵子的甲酸和钼依赖性转录控制。对FhlA蛋白N端序列的分析揭示了一个独特的基序（氨基酸7-37），在与ABC型转运蛋白家族的几个成员相关的ATP酶中也发现了该基序。缺乏这些氨基酸的FhlA缺失衍生物（FhlA9-2）在体内无法激活hyc操纵子，尽管FhlA9-2在体外确实能与hyc启动子DNA结合。FhlA9-2-DNA-甲酸复合物的ATP酶活性比天然蛋白-DNA-甲酸复合物至少高三倍，并且在较低的甲酸水平下就能达到这种活性程度。将缺失延伸至氨基酸117（FhlA167）不仅逆转了FhlA9-2的FhlA（-）表型，还导致了对钼和甲酸的不依赖性。删除整个N端结构域（在692个氨基酸的蛋白质的氨基酸残基5和374之间）也导致了一种效应物非依赖性转录激活剂（FhlA165），其hyc操纵子表达水平比天然蛋白高两倍。FhlA165和FhlA167仍然需要ModE-Mo作为二级激活剂以实现hyc-lac表达的最佳水平。FhlA165蛋白对hyc启动子DNA的亲和力也比天然FhlA蛋白高两倍，而FhlA167蛋白在体外对hyc启动子DNA的亲和力显著降低。尽管天然蛋白的ATP酶活性因甲酸而增加，但FhlA165和FhlA167的ATP酶活性均对甲酸无反应。去除FhlA蛋白的前117个氨基酸似乎导致了编码甲酸氢裂解酶复合物组分的基因转录的组成型、效应物非依赖性激活。与ABC-ATP酶的序列相似性，结合FhlA缺失蛋白的特性表明，天然FhlA蛋白的N端区域与甲酸转运蛋白相互作用，既是甲酸转运促进剂又是细胞质受体。
