Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase.
Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.
Graentzdoerffer
Andrea
A
Institut für MikrobiologieMartin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06099, Halle, Germany.
Rauh
David
D
Pich
Andreas
A
Andreesen
Jan R
JR
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005051:Eubacterium / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D006864:Hydrogenase / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D008954:Models, Biological; D008957:Models, Genetic; D005810:Multigene Family; D011622:Pterins / Q000032:analysis / Q000302:isolation & purification; D012643:Selenium / Q000032:analysis*; D014158:Transcription, Genetic; D014414:Tungsten / Q000032:analysis*
D007506:Iron-Sulfur Proteins; D011622:Pterins; C118830:iron hydrogenase; D006864:Hydrogenase; D005560:Formate Dehydrogenases; D012643:Selenium; D014414:Tungsten