Eived and developed the experiments: QZ LY HX. Performed the experiments
Eived and made the experiments: QZ LY HX. Performed the experiments: QZ HC LY HX. Analyzed the information: QZ HC LY HX. Contributed reagents/materials/analysis tools: LY QZ. Wrote the manuscript: QZ.
NIH Public AccessAuthor ManuscriptBiochemistry. Author manuscript; out there in PMC 2014 October 28.Published in final edited type as: Biochemistry. 2013 April 30; 52(17): 2905913. doi:ten.1021/bi4003343.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptThe orphan protein bis–glutamylcystine reductase joins the pyridine nucleotide-disulfide reductase familyJuhan Kim1,2 and Shelley D. Copley1,two,*1Departmentof Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Boulder, Colorado 80309, United States2CooperativeInstitute for Investigation in Environmental Sciences, University of Colorado Boulder, Boulder, Colorado 80309, United StatesAbstractFacile DNA sequencing became doable decades after many enzymes had been JAK3 Inhibitor Accession purified and characterized. Consequently, you can find nevertheless “orphan” enyzmes whose activity is recognized but the genes that encode them have not been identified. Identification in the genes encoding orphan enzymes is vital because it enables correct annotation of genes of unknown function or with mis-assigned function. Bis–glutamylcystine reductase (GCR) is an orphan protein that was purified in 1988. This enzyme catalyzes the reduction of bis–glutamylcystine. Glutamylcysteine (-Glu-Cys) is definitely the big 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 from the protein predicted to be encoded by a gene in Halobacterium sp. NRC-1 that may be annotated as mercuric reductase. GCR and mercuric reductase activities had been assayed making use of 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 entire genome sequences are obtainable have close homologs of GCR, suggesting that there is much more to be learned concerning the low molecular weight thiols applied in halobacteria. Enormous genome sequencing efforts in current years have contributed millions of sequences to genomic databases. Functions for the vast majority of those sequences have already been predicted computationally primarily based upon sequence similarities to other proteins and a variety of other genomic clues like genome context and phylogenetic profiling.1 Computational annotations are usually correct in the superfamily level. On the other hand, predictions of certain functions are often wrong. Consequently of mis-annotation and subsequent transfer of erroneous annotations, the database is littered with incorrect assignments of function.4 On the other side in the picture, there are numerous “orphan” proteins for which functions are identified but for which the BRaf Inhibitor site corresponding genes haven’t been identified.5 Bis–*To whom correspondence needs to be addressed: Shelley D. Copley, Division of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado 80309, USA, Tel: (303) 492-6328, Fax: (303) 492-1149, [email protected]. Supplemental Materials may possibly be accessed totally free of charge on the net at pubs.acs.org.Kim and CopleyPageglutamylcystine reductase (GCR) is one of these orphan proteins. GCR from Halobacterium halobium was purified and c.