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, 52 (17), 2905-13

The Orphan Protein Bis-γ-Glutamylcystine Reductase Joins the Pyridine Nucleotide Disulfide Reductase Family

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The Orphan Protein Bis-γ-Glutamylcystine Reductase Joins the Pyridine Nucleotide Disulfide Reductase Family

Juhan Kim et al. Biochemistry.

Abstract

Facile DNA sequencing became possible decades after many enzymes had been purified and characterized. Consequently, there are still "orphan" enyzmes for which activities are known but for which encoding genes have not been identified. Identification of the genes encoding orphan enzymes is important because it allows correct annotation of genes of unknown function or with misassigned function. Bis-γ-glutamylcystine reductase (GCR) is an orphan protein that was purified in 1988. This enzyme catalyzes the reduction of bis-γ-glutamylcystine. γ-Glutamylcysteine is the major low-molecular weight thiol in halobacteria. We purified GCR from Halobacterium sp. NRC-1 and identified the sequence of 23 tryptic peptides by nano-liquid chromatography electrospray ionization tandem mass spectrometry. These peptides cover 62% of the protein predicted to be encoded by a gene in Halobacterium sp. NRC-1 that is annotated as mercuric reductase. GCR and mercuric reductase activities were assayed using enzyme that was expressed in Escherichia coli and refolded from inclusion bodies. The enzyme had robust GCR activity but no mercuric reductase activity. The genomes of most, but not all, halobacteria for which whole genome sequences are available have close homologues of GCR, suggesting that there is more to be learned about the low-molecular weight thiols used in halobacteria.

Figures

Figure 1
Figure 1
The reaction catalyzed by bis-γ-glutamylcystine reductase (GCR).
Figure 2
Figure 2
Mapping of peptides derived from tryptic digestion of Halobacterium sp. NRC-1 GCR onto the sequence of a protein annotated as mercuric reductase (accession number NP_279293). Fragments detected from ESI-MS/MS analysis are shown as dotted bars under the corresponding sequences. Fragments detected by ESI-MS/MS covered 287 of 465 amino acid residues.
Figure 3
Figure 3
(A) SDS-PAGE analysis of Halobacterium sp. NRC-1 N-His6-GCR overproduced in E. coli Arctic (DE3) RP. Lane 1, lysate; lane 2, soluble supernatant; lane 3, insoluble precipitate; lane 4, refolded protein; lane 5, protein obtained after purification using an immobilized Cu2+ resin. (B) GCR activity of the purified protein as a function of bis-γ-glutamylcystine concentration.
Figure 4
Figure 4
Pairwise sequence identities between selected proteins belonging to different families in the pyridine nucleotide disulfide reductase superfamily. Dihydrolipoamide dehydrogenase (DLDH), glutathione reductase (GR), bis-γ-glutamylcystine reductase (GCR), mycothione reductase (MYTR), trypanothione reductase (TYTR), and mercuric reductase (MERA). The box-and whisker plot shows the minimum, 1/4 quartile, median, average (●), 3/4 quartile, and maximum pairwise sequence identities between these sequences. The accession numbers for the proteins shown are listed in Supplemental Table 4.
Figure 5
Figure 5
Multiple sequence alignment of GCRs from several halobacteria with sequences from other subfamilies of the pyridine nucleotide disulfide reductase family, including dihydrolipoamide reductases (LPDA), glutathione reductases (GR), mycothione reductases (MYTR), trypanothione reductases (TYTR), and mercuric reductases (MERA). Residues found in conserved motifs involved with interactions with the flavin or NAD(P), catalytic cysteines and the C-terminal two-cysteine motif of the mercuric reductase are indicated above the alignment,. Accession numbers for the proteins shown are listed in Supplemental Table 4.
Figure 6
Figure 6
Major low-molecular-weight thiols used by diverse lineages mapped onto a phylogenetic tree constructed from 16S rRNA sequences (courtesy of Norman Pace). Note that CoA is used as a cofactor in all domains of life, but also serves the roles of typical low-molecular weight thiols in Staphylococcus, Bacillus, and Pyrodictium. Similarly, cysteine serves as the primary low-molecular-weight thiol in Giardia, Entamoeba and Trichomonas. References for the occurrence of other thiols; glutathione amide, mycothiol,, bacillithiol,, trypanothione and glutathionylspermidine, glutathionylspermidine in E. coli, ergothioneine, CoM-SS-CoB,, and CoM.,
Figure 7
Figure 7
Occurrence of γ-glutamylcysteine synthase (GshA) and bis-γ-glutamylcystine reductase (GCR) among halobacteria. The presence (●) or absence (□) of each protein is indicated for each species on the phylogenetic cladogram inferred from whole genome information using CVTree v.2.

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