A soluble NADH-dependent fumarate reductase in the reductive tricarboxylic acid cycle of Hydrogenobacter thermophilus TK-6

Abstract

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.

FIG. 1.

SDS-PAGE (13% acrylamide gel) of purified FRD (A) and native PAGE (7.5% acrylamide gel) of FRD solutions from late purification steps (B). (A) Lane 1, molecular mass markers (each band was 0.40 μg protein); lane 2, purified FRD (total, 1.5 μg protein). (B) Lane 1, DEAE fraction; lane 2, hydroxyapatite fraction; lane 3, MonoQ fraction; lane 4, purified FRD. Equivalent amounts of FRD activity were applied for all fractions.

FIG. 2.

Phylogenetic trees of FRD/SDH catalytic subunits (A) and iron-sulfur subunits (B). The numbers at the nodes are bootstrap confidence values expressed as percentages of 1,000 bootstrap replications. The order of divergence was presumed to be reliable only when the bootstrap values were above 80. The scale bars represent 0.2 estimated change per nucleotide. The trees were constructed by using the neighbor-joining method and showed the same overall topology as the trees constructed by the maximum likelihood method. The organism, gene, and locus tag in the NCBI protein database (in parentheses) for the sequences used in these figures are as follows: for panel A, Acidianus ambivalens sdhA (CAA06780), A. aeolicus aq_594 (NP_213415), Bacillus subtilis sdhA (P08065), Campylobacter jejuni frdA (NP_281599), E. coli frdA and sdhA (Escherichia 1 and 2; AAC77114 and AAC73817, respectively), Geobacter sulfurreducens frdA (NP_952230), Helicobacter pylori frdA (NP_222899), Hydrogenivirga sp. strain 128-5-R1-1 HG1285_17779 (ZP_02177006), H. thermophilus frdA (AB437311), Hydrogenobaculum sp. strain Y04AAS1 HY04AAS1DRAFT_0051 (ZP_02060827), Magnetococcus sp. strain MC-1 Mmc1_1746 (YP_865660), M. thermoautotrophicus tfrA (CAA04398), Nitratiruptor sp. strain SB155-2 NIS_0006 (YP_001355481), Paenibacillus macerans sdhA (CAA69872), Pyrobaculum islandicum Pisl_0247 (ABL87427), S. cerevisiae FRDS, OSM1, and SDH1 (Saccharomyces 1, 2, and 3; AAB64995, AAB59346, and AAA35026, respectively), Shewanella frigidimarina fccA (P0C278), Sulfolobus acidocaldarius sdhA (AAY80343), Sulfurimonas denitrificans Suden_0037 (YP_392553), Sulfurovum sp. strain NBC37-1 SUN_0152 (YP_001357469), Thermoproteus neutrophilus Tneu_0423 (ACB39371), T. brucei frdg and frdm1 (Trypanosoma 1 and 2; AAN40014 and AAX20163, respectively), and Wolinella succinogenes frdA (P17412); for panel B, A. ambivalens sdhB (CAA06781), A. aeolicus aq_553 (NP_213388), B. subtilis sdhB (P08066), C. jejuni frdB (NP_ 281600), E. coli frdB and sdhB (Escherichia 1 and 2; AAC77113 and AAC73818, respectively), G. sulfurreducens frdB (NP_952231), H. thermophilus frdB (AB437312), M. thermoautotrophicus tfrB (CAA04399), Nitratiruptor sp. strain SB155-2 NIS_0010 (YP_001355485), P. macerans sdhB (CAA69873), P. islandicum Pisl_0246 (ABL87426), S. cerevisiae SDH2 (AAS56515), S. acidocaldarius sdhB (AAY80342), S. denitrificans Suden_0038 (YP_392554), T. neutrophilus Tneu_0424 (ACB39372), and W. succinogenes frdB (P17596).

FIG. 3.

Alignment of amino acid sequences of catalytic subunits. The organism and gene for the sequences used in this figure are as follows: Ht, H. thermophilus frdA; Ec, E. coli frdA and sdhA; Ws, Wolinella succinogenes frdA; Sc, S. cerevisiae SDH1, FRDS, and OSM1; Sf, Shewanella frigidimarina fccA; Tb, T. brucei frdg and frdm1; Mt, M. thermoautotrophicus tfrA. The conserved histidine (His42 in H. thermophilus) and the “GG doublet” (49) are shown with a gray and a white box, respectively.

FIG. 4.

Phylogenetic tree of H. thermophilus FrdC and its homologous sequences in members of the Aquificaceae, with the catalytic subunits of molybdenum enzymes and NuoG. The numbers at the nodes are bootstrap confidence values expressed as percentages of 1,000 bootstrap replications. The order of divergence was presumed to be reliable only when the bootstrap values were above 80. The scale bar represents 0.5 estimated change per nucleotide. The tree was constructed by using the neighbor-joining method and showed the same overall topology as the tree constructed by the maximum likelihood method. The organism, gene, and locus tag in the NCBI protein database (in parentheses) for the sequences used in this figure are as follows: A. aeolicus aq_116 (NP_ 213078), Buchnera aphidicola subsp. Baizongia pistaciae nuoG (NP_777777), Escherichia coli K-12 strain MG1655 dmsA, fdhF, fdnG, and narG and Escherichia coli O157:H7 nuoG (Escherichia 1, 2, 3, 4, 5; AAC73980, AAD13462, AAD13438, AAC74308, and Q8XCX2, respectively), Haloarcula marismortui narG (CAB89111), Haloferax mediterranei narG and nasA (Haloferax 1 and 2; CAF21906 and CAF19042, respectively), Hydrogenivirga sp. strain 128-5-R1-1 HG1285_08874 (ZP_02178505), H. thermophilus frdC (AB437313), Hydrogenobaculum sp. strain Y04AAS1 HY04AAS1DRAFT_0487 (ZP_02060585), Klebsiella pneumoniae nasA (B49847), Methanobacterium formicicum fdhA (J02581), Mycobacterium tuberculosis nuoG (NP_337764), Pseudomonas aeruginosa nuoG and Pseudomonas putida narB (Pseudomonas 1 and 2; AAG06030 and AAF74559, respectively), Rhodobacter capsulatus dorA and nasA (Rhodobacter 1 and 2; Q52675 and AAQ18186, respectively), Shewanella oneidensis nuoG (NP_716644), Sinorhizobium meliloti nuoG2 (AAK65486), Streptomyces coelicolor nuoG (NP_628730), and Thermus thermophilus nqo3 (Q56223). DMSO, dimethyl sulfoxide.