Two Tandemly Arranged Ferredoxin Genes in the<i>Hydrogenobacter thermophilus</i>Genome: Comparative Characterization of the Recombinant [4Fe–4S] Ferredoxins

Ikeda, Takeshi; Yamamoto, Masahiro; Arai, Hiroyuki; Ohmori, Daijiro; Ishii, Masaharu; Igarashi, Yasuo

doi:10.1271/bbb.69.1172

Cited by 16 publications

(31 citation statements)

References 20 publications

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“…Previous reports have suggested some evidence of gene duplication, rearrangement, fusion, and loss on the chromosomal DNA of strain TK-6 (Ikeda et al, 2005). Approximately 1.0 Â 10 3 transformants were obtained by a transformation using 10 mg of plasmid DNA and 2.8 Â 10 8 of competent cells.…”

Section: Resultsmentioning

confidence: 99%

Role of two 2-oxoglutarate:ferredoxin oxidoreductases inHydrogenobacter thermophilusunder aerobic and anaerobic conditions

Yamamoto¹,

Arai²,

Ishii³

et al. 2006

FEMS Microbiology Letters

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Hydrogenobacter thermophilus TK-6 is a thermophilic, hydrogen-oxidizing bacterium that fixes carbon dioxide as a sole carbon source via the reductive tricarboxylic acid cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is one of the key enzymes in the pathway. Strain TK-6 has at least two isozymes of OGOR, namely For and Kor. These OGORs showed different expression patterns under aerobic conditions than under anaerobic conditions. In this work, we developed a homologous recombination method for Hydrogenobacter, and constructed a For mutant and a Kor mutant. Observation of phenotypes of the mutant strains showed that Kor was essential for anaerobic growth and that For activity supported robust aerobic growth of the organism.

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Section: Resultsmentioning

confidence: 99%

Role of two 2-oxoglutarate:ferredoxin oxidoreductases inHydrogenobacter thermophilusunder aerobic and anaerobic conditions

Yamamoto¹,

Arai²,

Ishii³

et al. 2006

FEMS Microbiology Letters

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“…When the production of succinate was measured, the reaction mixtures contained 50 mM NaPO 4 (pH 6.5), 5 or 20 mM fumarate, 0.5 mM NADH or 5 mM MV, and the enzyme solution in a total volume of 500 l. To analyze FRD activity with FADH 2 , FMNH 2 , BV, and ferredoxins, 0.25 mM FAD, 0.25 mM FMN, 10 mM BV, and 10 M ferredoxins were used, respectively. Ferredoxins were heterologously expressed and purified as previously described (25,28). MV, BV, FADH 2 , FMNH 2 , and ferredoxins were reduced with 5 or 10 mM dithionite (28).…”

Section: Methodsmentioning

confidence: 99%

“…When NADPH, FADH 2 , or FMNH 2 was used as an electron donor, no significant activity was detected. Also, several enzymes in the RTCA cycle are known to react with ferredoxins, and several ferredoxins have been found in H. thermophilus (25,28). Therefore, the reactivity with these ferredoxins was examined, but no ferredoxin-dependent FRD activity was detected.…”

Section: Purification Of Frd From H Thermophilusmentioning

confidence: 99%

A Soluble NADH-Dependent Fumarate Reductase in the Reductive Tricarboxylic Acid Cycle of Hydrogenobacter thermophilus TK-6

et al. 2008

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Fumarate reductase (FRD) is an enzyme that reduces fumarate to succinate. In many organisms, it is bound to the membrane and uses electron donors such as quinol. In this study, an FRD from a thermophilic chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6, was purified and characterized. FRD activity using NADH as an electron donor was not detected in the membrane fraction but was found in the soluble fraction. The purified enzyme was demonstrated to be a novel type of FRD, consisting of five subunits. One subunit showed high sequence identity to the catalytic subunits of known FRDs. Although the genes of typical FRDs are assembled in a cluster, the five genes encoding the H. thermophilus FRD were distant from each other in the genome. Furthermore, phylogenetic analysis showed that the H. thermophilus FRD was located in a distinct position from those of known soluble FRDs. This is the first report of a soluble NADH-dependent FRD in Bacteria and of the purification of a FRD that operates in the reductive tricarboxylic acid cycle.

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“…Three ferredoxins of H. thermophilus, namely, Fd1, Fd2, and Fd3, were expressed in E. coli and purified by heat treatment, Q Sepharose fast-flow chromatography, and Superdex 75 HR 10/30 chromatography as previously described (8). Fd3 was further purified on a MonoQ column; the Superdex 75 fractions containing Fd3 were pooled and loaded onto a MonoQ column equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 1 mM MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

“…Fd-GOGATs from phototrophs are reported to react with [2Fe-2S] ferredoxins (13). Two [4Fe-4S] ferredoxins (Fd1 and Fd2) have been found in H. thermophilus, and they were purified from recombinant E. coli (8). From the draft genome data, a novel ferredoxin gene, fdx3 (encoding Fd3), was found in the genome of H. thermophilus.…”

Section: Vol 189 2007 Ferredoxin-type Glutamate Synthase Of H Thermentioning

confidence: 99%

A Novel Ferredoxin-Dependent Glutamate Synthase from the Hydrogen-Oxidizing Chemoautotrophic BacteriumHydrogenobacter thermophilusTK-6

et al. 2007

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Glutamate synthases are classified according to their specificities for electron donors. Ferredoxin-dependent glutamate synthases had been found only in plants and cyanobacteria, whereas many bacteria have NADPHdependent glutamate synthases. In this study, Hydrogenobacter thermophilus, a hydrogen-oxidizing chemoautotrophic bacterium, was shown to possess a ferredoxin-dependent glutamate synthase like those of phototrophs. This is the first observation, to our knowledge, of a ferredoxin-dependent glutamate synthase in a nonphotosynthetic organism. The purified enzyme from H. thermophilus was shown to be a monomer of a 168-kDa polypeptide homologous to ferredoxin-dependent glutamate synthases from phototrophs. In contrast to known ferredoxin-dependent glutamate synthases, the H. thermophilus glutamate synthase exhibited glutaminase activity. Furthermore, this glutamate synthase did not react with a plant-type ferredoxin (Fd3 from this bacterium) containing a [2Fe-2S] cluster but did react with bacterial ferredoxins (Fd1 and Fd2 from this bacterium) containing [4Fe-4S] clusters. Interestingly, the H. thermophilus glutamate synthase was activated by some of the organic acids in the reductive tricarboxylic acid cycle, the central carbon metabolic pathway of this organism. This type of activation has not been reported for any other glutamate synthases, and this property may enable the control of nitrogen assimilation by carbon metabolism.Glutamate synthase (glutamine:2-oxoglutarate amidotransferase [GOGAT]) is known to be one of the most important enzymes for ammonium assimilation. This enzyme synthesizes Glu from Gln and 2-oxoglutarate (2-OG) as follows: Gln ϩ 2-OG ϩ reduced electron carrier 3 2 Glu ϩ oxidized electron carrier.GOGAT couples with glutamine synthetase (GS) for ammonium assimilation, and their total reaction incorporates NH 3 into 2-OG at the expense of ATP and reducing power. This coupling reaction is called the GS/GOGAT pathway, a prevailing ammonium assimilatory pathway among many organisms (15,22).GOGATs are classified into several types according to their specificities for electron donors (27). The first type is ferredoxindependent GOGAT (Fd-GOGAT), which utilizes a reduced ferredoxin as an electron donor. Because Fd-GOGAT is found only in cyanobacteria and chloroplasts of plants and is not found in nonphotosynthetic organisms, it is often called "planttype GOGAT." Another type of GOGAT utilizes NADPH as an electron donor. NADPH-dependent GOGAT (NADPH-GOGAT) is common among most bacteria and is often referred to as "bacterial GOGAT."Fd-GOGAT is a monomeric protein with a molecular mass of Ϸ150 kDa, while NADPH-GOGAT forms an (␣␤) 4 heterooctamer with ␣ subunits of Ϸ150 kDa and ␤ subunits of Ϸ50 kDa. Fd-GOGAT and the ␣ subunit of NADPH-GOGAT are homologous to each other. There is an insertion of Ϸ18 amino acid residues conserved in Fd-GOGATs from phototrophs but not in reported NADPH-GOGATs. This insertion is called the Fd loop and is suspected to be involved in ferredoxin-Fd-GOGAT binding (26,27,28...

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Two Tandemly Arranged Ferredoxin Genes in theHydrogenobacter thermophilusGenome: Comparative Characterization of the Recombinant [4Fe–4S] Ferredoxins

Cited by 16 publications

References 20 publications

Role of two 2-oxoglutarate:ferredoxin oxidoreductases inHydrogenobacter thermophilusunder aerobic and anaerobic conditions

Role of two 2-oxoglutarate:ferredoxin oxidoreductases inHydrogenobacter thermophilusunder aerobic and anaerobic conditions

A Soluble NADH-Dependent Fumarate Reductase in the Reductive Tricarboxylic Acid Cycle of Hydrogenobacter thermophilus TK-6

A Novel Ferredoxin-Dependent Glutamate Synthase from the Hydrogen-Oxidizing Chemoautotrophic BacteriumHydrogenobacter thermophilusTK-6

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