title: N-terminal truncations in the FhlA protein result in formate- and MoeA-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli.
abstract: 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.
authors.affiliation: Department of Microbiology and Cell Science, Box 110700, University of Florida, Gainesville, FL 32611, USA.
authors.forename: W T
authors.initials: WT
authors.lastname: Self
authors.forename: A
authors.initials: A
authors.lastname: Hasona
authors.forename: K T
authors.initials: KT
authors.lastname: Shanmugam
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D001426:Bacterial Proteins; D001665:Binding Sites; D004789:Enzyme Activation; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D005560:Formate Dehydrogenases / Q000235:genetics*; D005561:Formates / Q000494:pharmacology; D006864:Hydrogenase / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics; D008982:Molybdenum / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics*; D009154:Mutation; D009566:Nitrates / Q000378:metabolism; D009876:Operon; D010100:Oxygen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013466:Sulfurtransferases; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000187:drug effects / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005561:Formates; C491064:ModE protein, E coli; C101050:ModE protein, bacteria; D009097:Multienzyme Complexes; D009566:Nitrates; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C030544:formic acid; C067189:fhlA protein, E coli; C044659:molybdate; D008982:Molybdenum; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase; C111302:MoeA protein, E coli; D013466:Sulfurtransferases; D001616:beta-Galactosidase; D000251:Adenosine Triphosphatases; D010100:Oxygen