The archaeon Methanosarcina acetivorans contains a protein disulfide reductase with an iron-sulfur cluster

Abstract

Methanosarcina acetivorans, a strictly anaerobic methane-producing species belonging to the domain Archaea, contains a gene cluster annotated with homologs encoding oxidative stress proteins. One of the genes (MA3736) is annotated as a gene encoding an uncharacterized carboxymuconolactone decarboxylase, an enzyme required for aerobic growth with aromatic compounds by species in the domain Bacteria. Methane-producing species are not known to utilize aromatic compounds, suggesting that MA3736 is incorrectly annotated. The product of MA3736, overproduced in Escherichia coli, had protein disulfide reductase activity dependent on a C(67)XXC(70) motif not found in carboxymuconolactone decarboxylase. We propose that MA3736 be renamed mdrA (methanosarcina disulfide reductase). Further, unlike carboxymuconolactone decarboxylase, MdrA contained an Fe-S cluster. Binding of the Fe-S cluster was dependent on essential cysteines C(67) and C(70), while cysteines C(39) and C(107) were not required. Loss of the Fe-S cluster resulted in conversion of MdrA from an inactive hexamer to a trimer with protein disulfide reductase activity. The data suggest that MdrA is the prototype of a previously unrecognized protein disulfide reductase family which contains an intermolecular Fe-S cluster that controls oligomerization as a mechanism to regulate protein disulfide reductase activity.

FIG. 1.

Organization of the M. acetivorans MA4664/MA3734-MA3743 gene cluster and comparison to gene clusters in other Methanosarcina species. (A) The MA4664/MA3734-MA3743 gene organization shown in line a is the original annotation, and that shown in line b is the proposed annotation. MA4664/MA3734-MA3743 is compared to the following gene clusters from other sequenced methanogens: M. mazei Go1 MM0629 to MM0638, M. burtonii DSM 6242 Mbur2373 to Mbur2380, and M. barkeri strain Fusaro Mbar_A2452 to Mbar_A2454 and Mbar_A0252 to Mbar_A0250. The arrows indicate the gene direction and relative size and spacing. Homologous genes are indicated by the same pattern and are centered on MA3736 (indicated by the solid arrow). Genes indicated by asterisks in the M. mazei and M. barkeri gene clusters were missed in the original annotation and encode desulforedoxin (Dx) homologs similar to the MA4664 product. Mbur2378 and Mbur2379 encode homologs of flavodoxin and rubrerythrin, respectively. The genes in M. barkeri are not contiguous, as indicated by slashes. (B) RT-PCR analysis of the MA4664/MA3734-MA3743 gene cluster in M. acetivorans. Predicted RT-PCR products are indicated in panel A by lines under the genes and are labeled with roman numerals. Predicted RT-PCR product sizes are indicated in parentheses. The roman numerals above the gel lanes correspond to the predicted RT-PCR products. For lane IV′ the reaction was performed without reverse transcriptase.

FIG. 2.

Alignment of amino acid sequences of MdrA homologs and AhpD from M. tuberculosis. Identical amino acid residues are indicated by asterisks. The active site cysteines of AhpD that are conserved in the MdrA homologs (C₆₇ and C₇₀ in MdrA) are indicated by solid arrowheads, and additional conserved cysteines (C₃₉ and C₁₀₇ in MdrA) not found in AhpD are indicated by open arrowheads. Sequences were aligned using CLUSTAL W. MdrA, M. acetivorans C2A; MM0631, M. mazei Go1; Mbar_A2454, M. barkeri strain Fusaro; Mbur2375, M. burtonii DSM 6242; AhpD, M. tuberculosis.

FIG. 3.

Protein disulfide reductase activity of MdrA as determined by the insulin turbidimetric method. (A) DTT-dependent protein disulfide reductase activity of MdrA. The assay was carried out by addition of 0.33 mM DTT in 100 mM potassium phosphate (pH 7.0) containing 0.13 mM bovine insulin in the absence of MdrA (⧫) and in the presence of different concentrations of MdrA, including 2.5 μM (▪), 5 μM (▴), 7.5 μM (□), and 10 μM (□). (B) Lipoamide-dependent protein disulfide reductase activity of MdrA. The assay was carried out by addition of 0.5 mM NADH in 100 mM potassium phosphate (pH 7.0) containing 0.13 mM bovine insulin, 0.05 mM lipoamide, and 0.4 U bovine lipoamide dehydrogenase in the absence of MdrA (⧫) and in the presence of different concentrations of MdrA, including 2.5 μM (▪), 5 μM (▴), 7.5 μM (⋄), and 10 μM (□). The insets show the linear dependence of the activity on the MdrA concentration.

FIG. 4.

Protein disulfide reductase activities of wild-type MdrA and cysteine variants. (A) DTT-dependent activity. (B) Lipoamide-dependent activity. Assays were performed as described in Materials and Methods. The values are reported as ΔA₆₅₀/min², 10⁻⁵. WT, wild type.

FIG. 5.

UV-visible spectra of wild-type MdrA and variants. Line a, wild-type MdrA (400 μM); line b, C₃₉S/C₁₀₇S (200 μM); line c, C₆₇S/C₇₀S (400 μM). The inset shows vials containing the protein solutions.

FIG. 6.

Effect of EDTA on the oligomeric state of MdrA and cysteine variants as analyzed by size exclusion chromatography. (A) Elution profiles of wild-type MdrA as purified (black line) and with EDTA (light gray line) and of apo-MdrA (dark gray line). (B) Elution profiles of the C₆₇S/C₇₀S variant as purified (black line) and with EDTA (gray line). (C) Elution profiles of the C₃₉S/C₁₀₇S variant as purified (black line) and with EDTA (gray line). Dashed vertical line a indicates the volume corresponding to the hexameric form of MdrA, and dashed vertical line b indicates the volume corresponding to the trimeric form of MdrA. Hexameric and trimeric volumes were calculated based on a standard curve generated with molecular mass standards (data not shown).

FIG. 7.

Phylogenetic tree of selected CMD-, MdrA-, and AhpD-related sequences. The phylogenetic tree was constructed using the neighbor-joining method. The scale bar indicates the average number of amino acid substitutions per site. Prototypical functionally analyzed CMD and AhpD, as well as MdrA, are in bold type. Cluster I contains AhpD-related proteins, and cluster II contains MdrA-related proteins. Cluster I and II proteins contain a CXXC motif, with the exception of TTHA0727 from T. thermophilus, which contains an SXXC motif (indicated by an asterisk). Cluster III contains prototypical CMD-related proteins that do not contain a CXXC motif.