title: Details in the catalytic mechanism of mammalian thioredoxin reductase 1 revealed using point mutations and juglone-coupled enzyme activities.
abstract: The mammalian selenoprotein thioredoxin reductase 1 (TrxR1) is a key enzyme in redox regulation, antioxidant defense, and cellular growth. TrxR1 can catalyze efficient reduction of juglone (5-hydroxy-1,4-naphthoquinone; walnut toxin) in a reaction which, in contrast to reduction of most other substrates of TrxR1, is not dependent upon an intact selenocysteine (Sec, U) residue of the enzyme. Using a number of TrxR1 mutant variants, we here found that a sole Cys residue at the C-terminal tail of TrxR1 is required for high-efficiency juglone-coupled NADPH oxidase activity of Sec-deficient enzyme, occurring with mixed one- and two-electron reactions producing superoxide. The activity also utilizes the FAD and the N-terminal redox active disulfide/dithiol motif of TrxR1. If a sole Cys residue at the C-terminal tail of TrxR1, in the absence of Sec, was moved further towards the C-terminal end of the protein compared to its natural position at residue 497, juglone reduction was, surprisingly, further increased. Ala substitutions of Trp407, Asn418 and Asn419 in a previously described "guiding bar", thought to mediate interactions of the C-terminal tail of TrxR1 with the FAD/dithiol site at the N-terminal domain of the other subunit in the dimeric enzyme, lowered turnover with juglone about 4.5-fold. Four residues of Sec-deficient TrxR1 were found to be easily arylated by juglone, including the Cys residue at position 497. Based upon our observations we suggest a model for involvement of the juglone-arylated C-terminal motif of TrxR1 to explain its high activity with juglone. This study thus provides novel insights into the catalytic mechanisms of TrxR1. One-electron juglone reduction by TrxR1 producing superoxide should furthermore contribute to the well-known prooxidant cytotoxicity of juglone.
authors.affiliation: School of Life Science and Medicine, Dalian University of Technology, Panjin 124221, China; Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden.
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Xu
authors.affiliation: Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden.
authors.forename: Qing
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden. Electronic address: Elias.Arner@ki.se.
authors.forename: Elias S J
authors.initials: ES
authors.lastname: Arnér
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D019255:NADPH Oxidases / Q000378:metabolism; D009285:Naphthoquinones / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D017354:Point Mutation; D017279:Selenocysteine / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013481:Superoxides / Q000378:metabolism; D054481:Thioredoxin Reductase 1 / Q000737:chemistry / Q000235:genetics*
chemical_list: D000596:Amino Acids; D009285:Naphthoquinones; D017279:Selenocysteine; D013481:Superoxides; D019255:NADPH Oxidases; C516124:TXNRD1 protein, human; D054481:Thioredoxin Reductase 1; D003545:Cysteine; C005134:juglone