title: 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 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.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology and Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder, Boulder, CO 80309, USA.
authors.forename: Juhan
authors.initials: J
authors.lastname: Kim
authors.forename: Shelley D
authors.initials: SD
authors.lastname: Copley
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002853:Chromatography, Liquid; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006217:Halobacterium / Q000201:enzymology; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D017931:DNA Primers; D009247:NADH, NADPH Oxidoreductases; C056781:bis-gamma-glutamylcystine reductase; C014703:disulfide reductase (NADH)
references.citation: Biochemistry. 2010 May 25;49(20):4374-82
references.pmid: 20415463
references.citation: PLoS Comput Biol. 2009 Dec;5(12):e1000605
references.pmid: 20011109
references.citation: Proteins. 1994 Feb;18(2):161-73
references.pmid: 8159665
references.citation: J Bacteriol. 2011 Sep;193(18):4904-13
references.pmid: 21764916
references.citation: Science. 2007 Sep 28;317(5846):1903-6
references.pmid: 17901330
references.citation: Biochemistry. 2005 Aug 2;44(30):10360-8
references.pmid: 16042413
references.citation: Drug Metab Rev. 2011 May;43(2):138-51
references.pmid: 21428697
references.citation: J Am Chem Soc. 2010 May 19;132(19):6632-3
references.pmid: 20420449
references.citation: Pharmacol Ther. 1990;47(1):61-71
references.pmid: 2195557
references.citation: Nucleic Acids Res. 2009 Jul;37(Web Server issue):W174-8
references.pmid: 19398429
references.citation: PLoS One. 2012;7(4):e34302
references.pmid: 22485162
references.citation: Biochemistry. 1999 Sep 7;38(36):11827-33
references.pmid: 10512639
references.citation: Res Microbiol. 2006 Jan-Feb;157(1):57-68
references.pmid: 16431085
references.citation: Nucleic Acids Res. 2010 Jan;38(Database issue):D211-22
references.pmid: 19920124
references.citation: Biochem J. 1995 Dec 1;312 ( Pt 2):465-9
references.pmid: 8526857
references.citation: FEBS J. 2005 Mar;272(5):1189-200
references.pmid: 15720393
references.citation: Adv Enzymol Relat Areas Mol Biol. 1991;64:1-53
references.pmid: 1675828
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Biochemistry. 2007 Mar 20;46(11):3234-45
references.pmid: 17323930
references.citation: Trends Genet. 2000 Jun;16(6):276-7
references.pmid: 10827456
references.citation: Proc Natl Acad Sci U S A. 2010 Apr 6;107(14):6482-6
references.pmid: 20308541
references.citation: Eur J Biochem. 2002 Apr;269(7):1895-904
references.pmid: 11952791
references.citation: Carcinogenesis. 1983 Sep;4(9):1097-1100
references.pmid: 6883632
references.citation: J Bacteriol. 1988 Aug;170(8):3459-67
references.pmid: 3136140
references.citation: Open Microbiol J. 2008;2:13-7
references.pmid: 19088906
references.citation: Bioeng Bugs. 2010 Jul-Aug;1(4):288-90
references.pmid: 21327063
references.citation: J Bacteriol. 1996 Apr;178(7):1990-5
references.pmid: 8606174
references.citation: Biotechnol Bioeng. 1999 Jul 5;64(1):38-45
references.pmid: 10397837
references.citation: BMC Bioinformatics. 2007 Jul 10;8:244
references.pmid: 17623104
references.citation: J Biol Chem. 2001 Jun 15;276(24):20890-7
references.pmid: 11399772
references.citation: Genome Biol. 2002;3(5):research0025
references.pmid: 12049666
references.citation: Science. 1988 Nov 11;242(4880):939-41
references.pmid: 3187533
references.citation: J Bacteriol. 2000 May;182(9):2629-34
references.pmid: 10762269
references.citation: Nucleic Acids Res. 1994 Sep;22(17):3590-6
references.pmid: 7937065
references.citation: Biochemistry. 1992 Feb 18;31(6):1677-85
references.pmid: 1531297
references.citation: J Bacteriol. 1985 Jan;161(1):438-41
references.pmid: 2857165
references.citation: J Biol Chem. 1989 Jan 15;264(2):719-25
references.pmid: 2910862
references.citation: Nat Chem Biol. 2009 Sep;5(9):625-7
references.pmid: 19578333
references.citation: Biochim Biophys Acta. 2013 May;1830(5):3182-98
references.pmid: 23075826
references.citation: Mol Biochem Parasitol. 2001 Jul;115(2):189-98
references.pmid: 11420105
references.citation: J Bacteriol. 2011 Apr;193(8):1981-90
references.pmid: 21335456
references.citation: Fungal Genet Biol. 2012 Feb;49(2):160-72
references.pmid: 22209968
references.citation: Biochemistry. 2011 Jun 28;50(25):5555-7
references.pmid: 21604715
references.citation: Bioinformatics. 2001 Sep;17(9):847-8
references.pmid: 11590104
references.citation: Biochemistry (Mosc). 2005 Nov;70(11):1199-211
references.pmid: 16336178
references.citation: Antioxid Redox Signal. 2006 May-Jun;8(5-6):753-62
references.pmid: 16771667
references.citation: J Am Chem Soc. 2004 Jul 14;126(27):8378-9
references.pmid: 15237985
references.citation: Nucleic Acids Res. 2010 Jan;38(Database issue):D161-6
references.pmid: 19858104
references.citation: Bioinformatics. 2007 Jul 1;23(13):i205-11
references.pmid: 17646298
references.citation: Nature. 2011 Jun 19;476(7360):341-5
references.pmid: 21685886
references.citation: Biochemistry. 2006 Sep 26;45(38):11278-89
references.pmid: 16981688
references.citation: J Biol Chem. 1982 Mar 10;257(5):2498-503
references.pmid: 6277900
references.citation: Nucleic Acids Res. 2010 Jul;38(Web Server issue):W695-9
references.pmid: 20439314
references.citation: Cell Mol Life Sci. 2006 Mar;63(5):517-23
references.pmid: 16465439
references.citation: Antioxid Redox Signal. 2013 May 1;18(13):1642-53
references.pmid: 23075082
references.citation: PLoS Comput Biol. 2007 Jul;3(7):e121
references.pmid: 17658942
references.citation: Trends Biochem Sci. 2009 Feb;34(2):85-96
references.pmid: 19135374
references.citation: Nucleic Acids Res. 2004 Mar 19;32(5):1792-7
references.pmid: 15034147