Semisynthesis and Characterization of Mammalian Thioredoxin Reductase

Eckenroth, B.E.; Harris, Katharine M.; Turanov, Anton A.; Gladyshev, Vadim N.; Raines, Ronald T.; Hondal, Robert J.

doi:10.1021/bi0517887

Cited by 78 publications

(130 citation statements)

References 47 publications

(76 reference statements)

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“…It was furthermore proposed that the C terminus of the enzyme had to form some type of ␤-turn-like bend to position the sulfur and selenium atoms of the two side chains close to each other (30). Several studies have subsequently involved modeling of this motif and tried to position it into the active site of the enzyme (32,33,(35)(36)(37), but its actual features in a crystal structure of the Seccontaining protein have not been determined until now. We are therefore able to analyze the features of this motif as found in the crystal structure, in view of the previous models and kinetic analyses, thereby probing the detailed molecular features in the catalytic cycle of rat TrxR1.…”

Section: Discussionmentioning

confidence: 99%

“…In 2001, the first crystal structure of a Sec-to-Cys mutant of rat TrxR1 was published (32), followed by several structure determinations of similar Sec-substituted mutants, i.e. mutants of mouse TrxR2 (33)(34)(35)(36), human TrxR1 (37) (also as directly deposited PDB entry 2CFY), and the non-selenoprotein orthologue of Drosophila (34,35). These structures as well as additional modeling studies and several extensive biochemical analyses (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) have generated a generally accepted model for the major features of the catalytic mechanism of mammalian TrxRs.…”

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confidence: 99%

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Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

Cheng

Sandalova

Lindqvist

et al. 2009

Journal of Biological Chemistry

182

183

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Selenoproteins contain a highly reactive 21st amino acid selenocysteine (Sec) encoded by recoding of a predefined UGA codon. Because of a lack of selenoprotein supply, high chemical reactivity of Sec, and intricate translation machineries, selenoprotein crystal structures are difficult to obtain. Structural prerequisites for Sec involvement in enzyme catalysis are therefore sparsely known. Here we present the crystal structure of catalytically active rat thioredoxin reductase 1 (TrxR1), revealing surprises at the C-terminal Sec-containing active site in view of previous literature. The oxidized enzyme presents a selenenylsulfide motif in trans-configuration, with the selenium atom of Sec-498 positioned beneath the side chain of Tyr-116, thereby located far from the redox active moieties proposed to be involved in electron transport to the Sec-containing active site. Upon reduction to a selenolthiol motif, the Sec residue moved toward solvent exposure, consistent with its presumed role in reduction of TrxR1 substrates or as target of electrophilic agents inhibiting the enzyme. A Y116I mutation lowered catalytic efficiency in reduction of thioredoxin, but surprisingly increased turnover using 5-hydroxy-1,4-naphthoquinone (juglone) as substrate. The same mutation also decreased sensitivity to inhibition by cisplatin. The results suggest that Tyr-116 plays an important role for catalysis of TrxR1 by interacting with the selenenylsulfide of oxidized TrxR1, thereby facilitating its reduction in the reductive half-reaction of the enzyme. The interaction of a selenenylsulfide with the phenyl ring of a tyrosine, affecting turnover, switch of substrate specificity, and modulation of sensitivity to electrophilic agents, gives important clues into the mechanism of TrxR1, which is a selenoprotein that plays a major role for mammalian cell fate and function. The results also demonstrate that a recombinant selenoprotein TrxR can be produced in high amount and sufficient purity to enable crystal structure determination, which suggests that additional structural studies of these types of proteins are feasible.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

Cheng

Sandalova

Lindqvist

et al. 2009

Journal of Biological Chemistry

182

183

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show abstract

“…to the C-terminus of mammalian thioredoxin reductase) [2], we decided to attempt oxidative deprotection using I 2 . Analytical HPLC analyses of these crude product mixtures resulted in extremely complex chromatograms.…”

Section: Model Cyclization Reaction Involving Sec(mob) and Cys(mob)mentioning

confidence: 99%

“…Our research efforts over the past several years have focused on synthesizing peptides containing selenocysteine (Sec, U) [1] (Abbreviations used in this paper follow the guidelines recommended by Jones JH) for use in peptide ligation reactions [2,3]. One problem with using Sec in solid-phase peptide synthesis (SPPS) is the requirement for using benzyl-type protecting groups for the selenol side-chain owing to the incompatibility of a trityl group (Trt) with this sidechain [4][5][6][7].…”

Section: Introductionmentioning

confidence: 99%

Studies on deprotection of cysteine and selenocysteine side‐chain protecting groups

Harris¹,

Flemer²,

Hondal³

2006

Journal of Peptide Science

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102

113

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Abstract:We present here a simple method for deprotecting p-methoxybenzyl groups and acetamidomethyl groups from the sidechains of cysteine and selenocysteine. This method uses the highly elecrophilic, aromatic disulfides 2,2 -dithiobis(5-nitropyridine) (DTNP) and 2,2 -dithiodipyridine (DTP) dissolved in TFA to effect removal of these heretofore difficult-to-remove protecting groups. The dissolution of these reagents in TFA, in fact, serves to 'activate' them for the deprotection reaction because protonation of the nitrogen atom of the pyridine ring makes the disulfide bond more electrophilic. Thus, these reagents can be added to any standard cleavage cocktail used in peptide synthesis.The p-methoxybenzyl group of selenocysteine is easily removed by DTNP. Only sub-stoichiometric amounts of DTNP are required to cause full removal of the p-methoxybenzyl group, with as little as 0.2 equivalents necessary to effect 70% removal of the protecting group. In order to remove the p-methoxybenzyl group from cysteine, 2 equivalents of DTNP and the addition of thioanisole was required to effect removal. Thioanisole was absolutely required for the reaction in the case of the sulfur-containing amino acids, while it was not required for selenocysteine. The results were consistent with thioanisole acting as a catalyst. The acetamidomethyl group of cysteine could also be removed using DTNP, but required the addition of >15 equivalents to be effective. DTP was less robust as a deprotection reagent. We also demonstrate that this chemistry can be used in a simultaneous cyclization/deprotection reaction between selenocysteine and cysteine residues protected by p-methoxybenzyl groups to form a selenylsulfide bond, demonstrating future high utility of the deprotection method.

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“…In addition, efficiency of Sec insertion into recombinant proteins is low, because the major products are often the truncated forms of selenoproteins. To overcome this problem, several methods have been proposed (33)(34)(35)(36), but none is fully satisfactory. Furthermore, some selenoproteins can be expressed only in eukaryotes because of unique posttranslational modifications.…”

Section: Development Of a Vector For Overexpression Of Selenoproteins Inmentioning

confidence: 99%

A highly efficient form of the selenocysteine insertion sequence element in protozoan parasites and its use in mammalian cells

Novoselov

Lobanov

Hua

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Selenoproteins are an elite group of proteins containing a rare amino acid, selenocysteine (Sec), encoded by the codon, UGA. In eukaryotes, incorporation of Sec requires a Sec insertion sequence (SECIS) element, a stem-loop structure located in the 3 -untranslated regions of selenoprotein mRNAs. Here we report identification of a noncanonical form of SECIS element in Toxoplasma gondii and Neospora canine, single-celled apicomplexan parasites of humans and domestic animals. This SECIS has a GGGA sequence in the SBP2-binding site in place of AUGA previously considered invariant. Using a combination of computational and molecular techniques, we show that Toxoplasma and Neospora possess both canonical and noncanonical SECIS elements. The GGGA-type SECIS element supported Sec insertion in mammalian HEK 293 and NIH 3T3 cells and did so more efficiently than the natural mammalian SECIS elements tested. In addition, mammalian type I and type II SECIS elements mutated into the GGGA forms were functional but manifested decreased Sec insertion efficiency. We carried out computational searches for both AUGA and GGGA forms of SECIS elements in Toxoplasma and detected five selenoprotein genes, including one coding for a previously undescribed selenoprotein, designated SelQ, and two containing the GGGA form of the SECIS element. In contrast, the GGGA-type SECIS elements were not detected in mammals and nematodes. As a practical outcome of the study, we developed pSelExpress1, a vector for convenient expression of selenoproteins in mammalian cells. It contains an SBP2 gene and the most efficient tested SECIS element: an AUGA mutant of the GGGA-type Toxoplasma SelT structure.genome ͉ RNA structure ͉ selenocysteine ͉ selenoprotein S elenocysteine (Sec)-containing proteins (selenoproteins) are rare but widely distributed in all domains of life (1), including bacteria (2, 3), archaea (4), and eukaryotes (5-7). The human genome possesses 25 genes encoding such proteins (7). The class of selenoproteins is defined by the occurrence of Sec, the 21st amino acid encoded by the UGA codon. Selenoproteins use the high reactivity of Sec, which is located in catalytic centers and serves redox function analogous to the functions of redox-active Cys residues (8). In addition to the UGA codon, a cis-acting element is present within selenoprotein genes, which is also essential for recognition of UGA as the Sec codon. This element is a stem-loop structure, known as the Sec insertion sequence (SECIS) located in coding regions of bacterial and in 3Ј-UTRs of archaeal and eukaryotic selenoprotein genes (9, 10).The principal features of the eukaryotic SECIS element include a segment containing four non-Watson-Crick base pairs UGAN. . . . NGAN (designated the quartet or core), an unpaired A preceding the quartet, and an unpaired AA or CC motif in the apical loop or bulge that is separated from the quartet by 11-12 nucleotides (11-14). Although having low sequence conservation, the secondary structure of eukaryotic SECIS elements is strictly conserved an...

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Semisynthesis and Characterization of Mammalian Thioredoxin Reductase

Cited by 78 publications

References 47 publications

Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

Studies on deprotection of cysteine and selenocysteine side‐chain protecting groups

A highly efficient form of the selenocysteine insertion sequence element in protozoan parasites and its use in mammalian cells

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