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.
Aldehyde oxidoreductase of Eubacterium acidaminophilum was purified to homogeneity under strict anaerobic conditions using a four‐step procedure. The purified enzyme was present as a monomer with an apparent molecular mass of 67 kDa and contained 6.0 ± 0.1 iron, 1.1 ± 0.2 tungsten, about 0.6 mol pterin cofactor and zinc, but no molybdenum. The enzyme activity was induced if a molar excess of electron donors, such as serine and/or formate, were supplied in the growth medium compared to readily available electron acceptors such as glycine betaine. Many aldehydes served as good substrates, thus enzyme activity obtained with acetaldehyde, propionaldehyde, butyraldehyde, isovaleraldehyde and benzaldehyde differed by a factor of less than two. Kinetic parameters were determined for all substrates tested. Oligonucleotides deduced from the N‐terminal amino acid sequence were used to isolate the encoding aorA gene and adjacent DNA regions. The deduced amino acid sequence of the aldehyde oxidoreductase exhibited high similarities to other tungsten‐containing aldehyde oxidoreductases from archaea. Transcription of the aorA gene was monocistronic and started from a σ54‐dependent promoter. Upstream of aorA, the gene aorR is localized whose product is similar to σ54‐dependent transcriptional activator proteins and, thus, AorR is probably involved in the regulation of aorA expression.
A probe based on the sequence of the gene encoding selenoprotein A of glycine reductase of Clostridium sticklandii was used to obtain clones of adjacent DNA that encoded the other components of glycine reductase, proteins B and C, in addition to thioredoxin and thioredoxin reductase. The genes of the thioredoxin system and the glycine reductase were shown to be transcribed together, confirming an operon structure. In addition, a gene (grdX) encoding a 13.7-kDa protein of unknown function seemed to be associated with the reductase genes. Four potential promoters were identified by mapping the 5'-end of the mRNAs. The sequence of promoter P1 was shown to be similar to the sigma70 promoter consensus sequence. The other three promoters were similar to each other, but not to known promoter consensus sequences. The transcripts starting at each of the four promoters were terminated to about 80% at a predicted loop structure downstream of grdB; the remaining transcripts continued through this structure and covered the genes encoding both subunits of protein C and bmpA, a gene that was also expressed monocistronically.
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