Role Of Selenocysteine Research Articles

The Key Role of Chalcogenurane Intermediates in the Reduction Mechanism of Sulfoxides and Selenoxides by Thiols Explored In Silico.

Sulfoxides and selenoxides oxidize thiols to disulfides while being reduced back to sulfides and selenides. While the reduction mechanism of sulfoxides to sulfides has been thoroughly explored experimentally as well as computationally, less attention has been devoted to the heavier selenoxides. In this work, we explore the reductive mechanism of dimethyl selenoxide, as an archetypal selenoxide and, for the sake of comparison, the reductive mechanism of dimethyl sulfoxide to gain insight into the role of the chalcogen on the reaction substrate. Particular attention is devoted to the key role of sulfurane and selenurane intermediates. Moreover, the capacity of these system to oxidize selenols rather than thiols, leading to the formation of selenyl sulfide bridges, is explored in silico. Notably, this analysis provides molecular insight into the role of selenocysteine in methionine sulfoxide reductase selenoenzyme. The activation strain model of chemical reactivity is employed in the studied reactions as an intuitive tool to bridge the computationally predicted effect of the chalcogen on the chalcogenoxide as well as on the chalcogenol.

Expression and purification of the membrane enzyme selenoprotein K

Selenoprotein K (SelK) is a membrane protein residing in the endoplasmic reticulum. The function of SelK is mostly unknown; however, it has been shown to participate in anti-oxidant defense, calcium regulation and in the endoplasmic reticulum associated protein degradation (ERAD) pathway. In order to study the function of SelK and the role of selenocysteine in catalysis, we have tested heterologous expression of human SelK in E. coli. Consequently, we have developed an over-expression strategy that exploits the maltose binding protein as a fusion partner to stabilize and solubilize SelK. The fusion partner can be cleaved from SelK in the presence of a variety of detergents compatible with structural characterization and the protein purified to homogeneity. SelK acquires a helical secondary structure in detergent micelles, even though it was predicted to be an intrinsically disordered protein due to its high percentage of polar residues. The same strategy was successfully applied to preparation of SelK binding partner – selenoprotein S (SelS). Hence, this heterologous expression and purification strategy can be applied to other members of the membrane enzyme family to which SelK belongs.

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.

The Role of Selenocysteine 133 in Catalysis by the Human Type 2 Iodothyronine Deiodinase

The Role of Selenocysteine 133 in Catalysis by the Human Type 2 Iodothyronine Deiodinase

The Role of Selenocysteine in ni, fe Hydrogenases: Biophysical and Synthetic Model Studies

The Role of Selenocysteine in ni, fe Hydrogenases: Biophysical and Synthetic Model Studies

The Role of Selenocysteine in Ni, Fe Hydrogenases: Biophysical and Synthetic Model Studies

The active site of Ni,Fe hydrogenases contains a heterodinuclear cluster where the Ni and Fe centers are bridged by two cysteine thiolate S-donor atoms. Two terminal cysteine thiolates are also present in the Ni coordination environment. In a few of the enzymes, one of the terminal thiolates is substituted by a selenocysteine. This substitution has an effect on the redox chemistry of the enzyme and on the catalytic mechanism. The presence of selenocysteine provides a spectroscopic probe of a specific Ni ligand. X-ray absorption spectroscopy was used to probe the Ni and Se environments in Desulfomicrobium baculatum hydrogenase in the oxidized and reduced states. These studies reveal that the Se environment is sensitive to the redox poise of the enzyme, while no changes in the Ni environment were observed. Crystallographically characterized model Ni complexes featuring alkylthiolate and alkylselenolate ligation are used explore the changes in structure and redox properties that might arise in the biological systems.