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

Abstract

Facile DNA sequencing became possible decades after many enzymes had been purified and characterized. Consequently, there are still “orphan” enyzmes whose activity is known but the genes that encode them 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 mis-assigned 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 (γ-Glu-Cys) 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 NanoLC 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 E. coli and re-folded 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 homologs of GCR, suggesting that there is more to be learned about the low molecular weight thiols used in halobacteria.

Iodotyrosine deiodinase: a unique flavoprotein present in organisms of diverse phyla

“…42,43 IYD revealed an origin that is much more ancient than would be anticipated by its iodide salvage activity in mammals and may indicate requirements for iodide or iodinated molecules in most multicellular organisms. The enzyme is now ready to join the ranks of other TH-associated proteins in describing the evolution of TH signaling.…”

“…The successful identification of a specialized enzyme such as IYD in diverse organisms demonstrates the importance of experimental data to support the functional assignment of a flavoprotein. 42,43 IYD revealed an origin that is much more ancient than would be anticipated by its iodide salvage activity in mammals and may indicate requirements for iodide or iodinated molecules in most multicellular organisms. The enzyme is now ready to join the ranks of other TH-associated proteins in describing the evolution of TH signaling.…”

Lateral Acquisitions Repeatedly Remodel the Oxygen Detoxification Pathway in Diplomonads and Relatives

“…GSH1 produces l -γ-glutamylcysteine, the only known cellular role of which is as a precursor of glutathione. The only known exception is that Halobacteria use l -γ-glutamylcysteine as an antioxidant through the enzyme bis-γ-glutamylcysteine reductase (Kim and Copley 2013). We did not detect homologs of this enzyme in any of the fornicata genomes.…”

Redox and Thiols in Archaea

“…Kim and Copley (2013) [33] further expressed an H. salinarum (sp. NRC-1) gene annotated as mercuric reductase (MerA) in Escherichia coli and demonstrated that the enzyme had robust NADPH-dependent GCR activity but no mercuric reductase activity [33]. The genomes of most, but not all, haloarchaea for which whole genome sequences are available have homologues that are at least 50% identical to GCR (UniRef 50 of UniProt Q9HSN0).…”

“…While the gene was not identified in this early work, Sundquist and Fahey provided evidence in H. salinarum for a γGC reductase (GCR) that was distinct from dihydrolipoamide dehydrogenase (DHD), the E3 component that oxidizes the thiol groups of dihydrolipoamide (Lip-(SH) 2 ) to lipoamide (Lip-(S) 2 ) in α-keto acid dehydrogenase complexes [31,32]. Kim and Copley (2013) [33] further expressed an H. salinarum (sp. NRC-1) gene annotated as mercuric reductase (MerA) in Escherichia coli and demonstrated that the enzyme had robust NADPH-dependent GCR activity but no mercuric reductase activity [33].…”