The conserved histidine 106 of large thioredoxin reductases is likely to have a structural role but not a base catalyst function

Jacob, Judit; Schirmer, R. Heiner; Gromer, Stephan

doi:10.1016/j.febslet.2005.01.001

Cited by 7 publications

(6 citation statements)

References 22 publications

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“…Recent findings indicate that H108 of large TrxR does not act as base-catalyst, supporting that our mutagenesis does not affect catalysis [27]. Fig.…”

Section: Resultssupporting

confidence: 86%

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Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

et al. 2006

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The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.

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“…Recent findings indicate that H108 of large TrxR does not act as base-catalyst, supporting that our mutagenesis does not affect catalysis [27]. Fig.…”

Section: Resultssupporting

confidence: 86%

“…Finally, H108 was changed to Y since the distance of the corresponding Y106 of hGR from GSSG is only $4.3 Å and allows hydrogen bonding with a cysteinyl residue thereof. Recent findings indicate that H108 of large TrxR does not act as base-catalyst, supporting that our mutagenesis does not affect catalysis [27]. Fig.…”

Section: Resultssupporting

confidence: 86%

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

et al. 2006

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“…While His 106 is conserved in TR there is a Thr substituted for Phe404′ for the mammalian enzymes (Thr437′ in 1ZKQ). His106 was previously suggested to be a base catalyst for DmTR (28) however results from recent mutagenesis studies indicate a structural role instead (31).…”

Section: Crystal Structure Of Tr From Drosophilamentioning

confidence: 97%

Structural and Biochemical Studies Reveal Differences in the Catalytic Mechanisms of Mammalian and Drosophila melanogaster Thioredoxin Reductases

et al. 2007

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Thioredoxin reductase (TR) from Drosophila melanogaster (DmTR) is a member of the glutathione reductase (GR) family of pyridine nucleotide disulfide oxidoreductases and catalyzes the reduction of the redox-active disulfide bond of thioredoxin. DmTR is notable for having high catalytic activity without the presence of a selenocysteine (Sec) residue (which is essential for the mammalian thioredoxin reductases). We report here the X-ray crystal structure of DmTR at 2.4 A resolution (Rwork = 19.8%, Rfree = 24.7%) in which the enzyme was truncated to remove the C-terminal tripeptide sequence Cys-Cys-Ser. We also demonstrate that tetrapeptides equivalent to the oxidized C-terminal active sites of both mouse mitochondrial TR (mTR3) and DmTR are substrates for the truncated forms of both enzymes. This truncated enzyme/peptide substrate system examines the kinetics of the ring-opening step that occurs during the enzymatic cycle of TR. The ring-opening step is 300-500-fold slower when Sec is replaced with Cys in mTR3 when using this system. Conversely, when Cys is replaced with Sec in DmTR, the rate of ring opening is only moderately increased (5-36-fold). Structures of these tetrapeptides were oriented in the active site of both enzymes using oxidized glutathione bound to GR as a template. DmTR has a more open tetrapeptide binding pocket than the mouse enzyme and accommodates the peptide Ser-Cys-Cys-Ser(ox) in a cis conformation that allows for the protonation of the leaving-group Cys by His464', which helps to explain why this TR can function without the need for Sec. In contrast, mTR3 shows a narrower pocket. One possible result of this narrower interface is that the mammalian redox-active tetrapeptide Gly-Cys-Sec-Gly may adopt a trans conformation for a better fit. This places the Sec residue farther away from the protonating histidine residue, but the lower pKa of Sec in comparison to that of Cys eliminates the need for Sec to be protonated.

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“…The precise mechanism of selenenylsulfide͞disulfide exchange has not yet been determined for mammalian TrxRs, but studies of D. melanogaster (DmTrxR1) (26,48,49) and P. falciparum (27,37,50) high-M r TrxRs provide considerable insight. DmTrxR1 exhibits significant sequence homology to mammalian TrxRs but is not a selenoprotein (26,48).…”

Section: Figmentioning

confidence: 99%

Crystal structures of oxidized and reduced mitochondrial thioredoxin reductase provide molecular details of the reaction mechanism

Biterova

Turanov

Gladyshev

et al. 2005

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

107

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Thioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. Through an intricate mechanism, these enzymes transfer reducing equivalents from NADPH to bound FAD and subsequently to an active-site disulfide. In mammalian TrxRs, the dithiol then reduces a mobile C-terminal selenocysteine-containing tetrapeptide of the opposing subunit of the dimer. Once activated, the C-terminal redox center reduces a disulfide bond within thioredoxin. In this report, we present the structural data on a mitochondrial TrxR, TrxR2 (also known as TR3 and TxnRd2). Mouse TrxR2, in which the essential selenocysteine residue had been replaced with cysteine, was isolated as a FAD-containing holoenzyme and crystallized (2.6 Å; R ‫؍‬ 22.2%; Rfree ‫؍‬ 27.6%). The addition of NADPH to the TrxR2 crystals resulted in a color change, indicating reduction of the active-site disulfide and formation of a species presumed to be the flavin-thiolate charge transfer complex. Examination of the NADP(H)-bound model (3.0 Å; R ‫؍‬ 24.1%; R free ‫؍‬ 31.2%) indicates that an active-site tyrosine residue must rotate from its initial position to stack against the nicotinamide ring of NADPH, which is juxtaposed to the isoalloxazine ring of FAD to facilitate hydride transfer. Detailed analysis of the structural data in conjunction with a model of the unusual C-terminal selenenylsulfide suggests molecular details of the reaction mechanism and highlights evolutionary adaptations among reductases.hioredoxins are the major cellular protein disulfide reductases and are responsible for the regulation of numerous biochemical processes within the cell (1). These proteins are maintained in a reduced state by thioredoxin reductases (TrxR), homodimeric f lavoproteins that catalyze the NADPHdependent reduction of thioredoxins (2, 3).Two forms of TrxRs have evolved with related but distinct modes of catalysis (2-5). Low-M r TrxRs (M r Ϸ 35 kDa) are typically found in prokaryotes, archaea, plants, and lower eukaryotes, whereas high-M r TrxRs (M r Ϸ 55 kDa) are observed in higher eukaryotes. To date, only the green algae Chlamydomonas reinhardtii has been shown to contain both a low-and a high-M r TrxR (6).The general features of catalysis are retained in both low-and high-M r TrxR (2, 4). TrxR transfers reducing equivalents from NADPH to its bound FAD, ultimately leading to the reduction of an active-site disulfide. In low-M r TrxRs, the catalytic cycle requires a large conformational change after dithiol activation (4,7,8). In high-M r TrxR, the active-site dithiol reduces a third redox active center in the highly mobile C terminus of the opposing subunit. This third group is responsible for the reduction of the disulfide bond within thioredoxin. Its nature is species-specific and ranges from a C-X-X-X-X-C disulfide in Plasmodium falciparum (9) to a vicinal disulfid...

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The conserved histidine 106 of large thioredoxin reductases is likely to have a structural role but not a base catalyst function

Cited by 7 publications

References 22 publications

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

Structural and Biochemical Studies Reveal Differences in the Catalytic Mechanisms of Mammalian and Drosophila melanogaster Thioredoxin Reductases

Crystal structures of oxidized and reduced mitochondrial thioredoxin reductase provide molecular details of the reaction mechanism

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