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.