Semisynthesis and characterization of mammalian thioredoxin reductase.

Biochemistry
Brian E EckenrothRobert J Hondal

Abstract

Thioredoxin reductase and thioredoxin constitute the cellular thioredoxin system, which provides reducing equivalents to numerous intracellular target disulfides. Mammalian thioredoxin reductase contains the rare amino acid selenocysteine. Known as the "21st" amino acid, selenocysteine is inserted into proteins by recoding UGA stop codons. Some model eukaryotic organisms lack the ability to insert selenocysteine, and prokaryotes have a recoding apparatus different from that of eukaryotes, thus making heterologous expression of mammalian selenoproteins difficult. Here, we present a semisynthetic method for preparing mammalian thioredoxin reductase. This method produces the first 487 amino acids of mouse thioredoxin reductase-3 as an intein fusion protein in Escherichia coli cells. The missing C-terminal tripeptide containing selenocysteine is then ligated to the thioester-tagged protein by expressed protein ligation. The semisynthetic version of thioredoxin reductase that we produce in this manner has k(cat) values ranging from 1500 to 2220 min(-)(1) toward thioredoxin and has strong peroxidase activity, indicating a functional form of the enzyme. We produced the semisynthetic thioredoxin reductase with a total yield of 24 mg fr...Continue Reading

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Citations

Apr 17, 2010·Amino Acids·Robert J Hondal, Erik L Ruggles
Nov 7, 2008·Biochemistry·Brian M LaceyRobert J Hondal
Dec 21, 2006·Journal of the American Chemical Society·Norman MetanisPhilip E Dawson
May 16, 2012·The Journal of General Physiology·Miguel Antonio AonSonia Cortassa
Dec 23, 2008·Antioxidants & Redox Signaling·Gloria Saab-Rincón, Brenda Valderrama
Nov 6, 2012·Antioxidants & Redox Signaling·Robert J HondalVadim N Gladyshev
May 2, 2007·Proceedings of the National Academy of Sciences of the United States of America·Sergey V NovoselovVadim N Gladyshev
Jun 28, 2011·Journal of Proteomics·Page C SpiessAlbert van der Vliet
May 2, 2009·Biochimica Et Biophysica Acta·Robert J Hondal
Jan 17, 2009·Free Radical Biology & Medicine·Olle RengbyElias S J Arnér
Apr 23, 2015·Bioorganic & Medicinal Chemistry·Lena SchmohlDirk Schwarzer
Mar 8, 2016·ACS Chemical Biology·Hans J Reich, Robert J Hondal
Oct 13, 2006·Journal of Peptide Science : an Official Publication of the European Peptide Society·Katharine M HarrisRobert J Hondal
Jun 13, 2008·Chembiochem : a European Journal of Chemical Biology·Giulio CasiDonald Hilvert
Nov 27, 2007·Journal of Peptide Science : an Official Publication of the European Peptide Society·Stevenson FlemerRobert J Hondal
Mar 18, 2016·Antioxidants & Redox Signaling·Dolph L Hatfield
Jul 6, 2012·Chembiochem : a European Journal of Chemical Biology·Watson J Lees
Jun 20, 2006·Biochemical and Biophysical Research Communications·Brian M Lacey, Robert J Hondal
Feb 28, 2013·Biophysical Journal·Jackelyn M KembroSonia Cortassa
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Feb 27, 2013·International Journal of Molecular Sciences·Ping ChenJia-Zuan Ni
Feb 17, 2018·Protein Science : a Publication of the Protein Society·N Connor PayneRobert J Hondal
Jul 28, 2018·Protein Science : a Publication of the Protein Society·Drew R Barber, Robert J Hondal
Jul 6, 2014·Physiological Reviews·Vyacheslav M LabunskyyVadim N Gladyshev
Dec 20, 2019·Journal of Peptide Science : an Official Publication of the European Peptide Society·Luis Moroder, Hans-Jürgen Musiol
Dec 5, 2008·The Journal of Biological Chemistry·Qing ChengElias S J Arnér
Sep 1, 2017·Angewandte Chemie·Reem MousaNorman Metanis
Apr 30, 2017·Chemical Science·L DeryN Metanis
Oct 30, 2020·Chemical Communications : Chem Comm·Toshiki TakeiHironobu Hojo
Nov 28, 2019·Chemical Reviews·Robert E Thompson, Tom W Muir
Jan 8, 2014·Biochemistry·Adam P LothropRobert J Hondal

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