Overexpression of wild type and SeCys/Cys mutant of human thioredoxin reductase in E. coli: the role of selenocysteine in the catalytic activity.
In contrast to Escherichia coli and yeast thioredoxin reductases, the human placental enzyme contains an additional redox center consisting of a cysteine-selenocysteine pair that precedes the C-terminal glycine residue. This reactive selenocysteine-containing center imbues the enzyme with its unusually wide substrate specificity. For expression of the human gene in E. coli, the sequence corresponding to the SECIS element required for selenocysteine insertion in E. coli formate dehydrogenase H was inserted downstream of the TGA codon in the human thioredoxin reductase gene. Omission of this SECIS element from another construct resulted in termination at UGA. Change of the TGA codon to TGT gave a mutant enzyme form in which selenocysteine was replaced with cysteine. The three gene products were purified using a standard isolation protocol. Binding properties of the three proteins to the affinity resins used for purification and to NADPH were similar. The three proteins occurred as dimers in the native state and exhibited characteristic thiolate-flavin charge transfer spectra upon reduction. With DTNB as substrate, compared to native rat liver thioredoxin reductase, catalytic activities were 16% for the recombinant wild type enzyme, about 5% for the cysteine mutant enzyme, and negligible for the truncated enzyme form.
Bar-Noy
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Laboratory of Biochemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-0320, USA.
Gorlatov
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Stadtman
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TC
D000818:Animals; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D019281:Dimerization; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006864:Hydrogenase / Q000235:genetics; D007700:Kinetics; D009097:Multienzyme Complexes / Q000235:genetics; D009154:Mutation; D009249:NADP / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012643:Selenium / Q000378:metabolism; D017279:Selenocysteine / Q000378:metabolism*; D012995:Solubility; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D003062:Codon; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D017279:Selenocysteine; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D004228:Dithionitrobenzoic Acid; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase; D013880:Thioredoxin-Disulfide Reductase; D012643:Selenium