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Semisynthesis and characterization of mammalian thioredoxin reductase.

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 from 6 L of E. coli culture (4 mg/L). This method allows production of a fully functional, semisynthetic selenoenzyme that is amenable to structure-function studies. A second semisynthetic system is also reported that makes use of peptide complementation to produce a partially active enzyme. The results of our peptide complementation studies reveal that a tetrapeptide that cannot ligate to the enzyme (Ac-Gly-Cys-Sec-Gly) can form a noncovalent complex with the truncated enzyme to form a weak complex. This noncovalent peptide-enzyme complex has 350-500-fold lower activity than the semisynthetic enzyme produced by peptide ligation.

SUBMITTER: Eckenroth B

PROVIDER: S-EPMC2570056 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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Semisynthesis and characterization of mammalian thioredoxin reductase.

Eckenroth Brian B Harris Katharine K Turanov Anton A AA Gladyshev Vadim N VN Raines Ronald T RT Hondal Robert J RJ

Biochemistry 20060401 16

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 m ...[more]

PMID: 16618105

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Project description:Mammalian thioredoxin reductase (TR) catalyzes the reduction of the redox-active disulfide bond of thioredoxin (Trx) and is similar in structure and mechanism to glutathione reductase except for a C-terminal 16-amino acid extension containing a rare vicinal selenylsulfide bond. This vicinal selenylsulfide bond is essentially a substrate for the enzyme's N-terminal redox center. Here we report the synthesis of peptide substrates for the truncated enzyme missing the C-terminal redox center. We developed a procedure for the synthesis of peptides containing cyclic vicinal disulfide/selenylsulfide bonds as well as their corresponding acyclic heterodimers. Vicinal disulfide bonds form eight-membered ring structures and are difficult to synthesize owing to their propensity to dimerize during oxidation. Our procedure makes use of two key improvements for on-resin disulfide bond formation presented previously by Galande and coworkers (Galande AK, Weissleder R, Tung C-H. An effective method of on-resin disulfide bond formation in peptides. J. Comb. Chem. 2005; 7: 174-177). First, the addition of an amine base to the deprotection solution allows the complete removal of the StBu group, allowing it to be replaced with a 5-Npys group. The second enhancement is the direct use of a Cys(Mob) or Sec(Mob) derivative as the nucleophilic partner instead of utilizing a naked sulfhydryl or selenol. These improvements result in the formation of a vicinal disulfide (or selenylsulfide) bond in high purity and yield. A direct comparison with the Galande procedure is presented. We also present a novel strategy for the formation of an acyclic, interchain selenylsulfide-linked peptide (linking H-PTVTGC-OH and H-UG-OH). Cysteine analogs of the cyclic and acyclic peptides were also synthesized. The results show that the ring structure contributes a factor of 52 to the rate, but the presence of selenium in the peptide is more important to catalysis than the presence of the ring.

Project description:High-molecular weight thioredoxin reductases (TRs) catalyze the reduction of the redox-active disulfide bond of thioredoxin, but an important difference in the TR family is the sequence of the C-terminal redox-active tetrapeptide that interacts directly with thioredoxin, especially the presence or absence of a selenocysteine (Sec) residue in this tetrapeptide. In this study, we have employed protein engineering techniques to investigate the C-terminal redox-active tetrapeptides of three different TRs: mouse mitochondrial TR (mTR3), Drosophila melanogaster TR (DmTR), and the mitochondrial TR from Caenorhabditis elegans (CeTR2), which have C-terminal tetrapeptide sequences of Gly-Cys-Sec-Gly, Ser-Cys-Cys-Ser, and Gly-Cys-Cys-Gly, respectively. Three different types of mutations and chemical modifications were performed in this study: insertion of alanine residues between the cysteine residues of the Cys-Cys or Cys-Sec dyads, modification of the charge at the C-terminus, and altering the position of the Sec residue in the mammalian enzyme. The results show that mTR3 is quite accommodating to insertion of alanine residues into the Cys-Sec dyad, with only a 4-6-fold drop in catalytic activity. In contrast, the activity of both DmTR and CeTR2 was reduced 100-300-fold when alanine residues were inserted into the Cys-Cys dyad. We have tested the importance of a salt bridge between the C-terminus and a basic residue that was proposed for orienting the Cys-Sec dyad of mTR3 for proper catalytic position by changing the C-terminal carboxylate to a carboxamide. The result is an enzyme with twice the activity as the wild-type mammalian enzyme. A similar result was achieved when the C-terminal carboxylate of DmTR was converted to a hydroxamic acid or a thiocarboxylate. Last, reversing the positions of the Cys and Sec residues in the catalytic dyad resulted in a 100-fold loss of catalytic activity. Taken together, the results support our previous model of Sec as the leaving group during reduction of the C-terminus during the catalytic cycle.

Project description:Cytosolic thioredoxin reductase 1 (TR1) is the best characterized of the class of high-molecular weight (Mr) thioredoxin reductases (TRs). TR1 is highly dependent upon the rare amino acid selenocysteine (Sec) for the reduction of thioredoxin (Trx) and a host of small molecule substrates, as mutation of Sec to cysteine (Cys) results in a large decrease in catalytic activity for all substrate types. Previous work in our lab and others has shown that the mitochondrial TR (TR3) is much less dependent upon the use of Sec for the reduction of small molecules. The Sec-dependent substrate utilization behavior of TR1 may be the exception and not the rule as we show that a variety of high-Mr TRs from other organisms, including Drosophila melanogaster, Caenorhabditis elegans, and Plasmodium falciparum, do not require Sec to reduce small molecule substrates, including 5,5'-dithiobis(2-nitrobenzoic acid), lipoic acid, selenite, and selenocystine. The data show that high-Mr TRs can be divided into two groups based upon substrate utilization patterns: a TR1 group and a TR3-like group. We have constructed mutants of TR3-like enzymes from mouse, D. melanogaster, C. elegans, and P. falciparum, and the kinetic data from these mutants show that these enzymes are less dependent upon the use of Sec for the reduction of substrates. We posit that the mechanistic differences between TR1 and the TR3-like enzymes in this study are due to the presence of a "guiding bar", amino acids 407-422, found in TR1, but not TR3-like enzymes. The guiding bar, proposed by Becker and co-workers [Fritz-Wolf, K., Urig, S., and Becker, K. (2007) The structure of human thioredoxin reductase 1 provides insights into C-terminal rearrangements during catalysis. J. Mol. Biol. 370, 116-127], restricts the motion of the C-terminal tail containing the C-terminal Gly-Cys-Sec-Gly, redox active tetrapeptide so that only this C-terminal redox center can be reduced by the N-terminal redox center, with the exclusion of most other substrates. This makes TR1 highly dependent upon the use of Sec because the selenium atom is responsible for both accepting electrons from the N-terminal redox center and donating them to the substrate in this model. Loss of both Se-electrophilicity and Se-nucleophilicity in the Sec → Cys mutant of TR1 greatly reduces catalytic activity. TR3-like enzymes, in contrast, are less dependent upon the use of Sec because the absence of the guiding bar in these enzymes allows for greater access of the substrate to the N-terminal redox center and because they can make use of alternative mechanistic pathways that are not available to TR1.

Dataset Information

Semisynthesis and characterization of mammalian thioredoxin reductase.

Publications

Semisynthesis and characterization of mammalian thioredoxin reductase.

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