Artificial Selenoenzyme Research Articles

Reversible Switch of Artificial Selenoenzyme Activity Modulated by Metal Ion-Induced Protein Nano-Assemblies

Reversible Switch of Artificial Selenoenzyme Activity Modulated by Metal Ion-Induced Protein Nano-Assemblies

Biomimetic Construction of Artificial Selenoenzymes.

Selenium exists in the form of selenocysteines in selenoproteins and plays a pivotal role in the catalytic process of the antioxidative enzymes. In order to study the structural and functional properties of selenium in selenoproteins, explore the significance of the role of selenium in the fields of biology and chemistry, scientists conducted a series of artificial simulations on selenoproteins. In this review, we sum up the progress and developed strategies in the construction of artificial selenoenzyme. Using different mechanisms from different catalytic angles, selenium-containing catalytic antibodies, semi-synthetic selenonezyme, and the selenium-containing molecularly imprinted enzymes have been constructed. A variety of synthetic selenoenzyme models have been designed and constructed by selecting host molecules such as cyclodextrins, dendrimers, and hyperbranched polymers as the main scaffolds. Then, a variety of selenoprotein assemblies as well as cascade antioxidant nanoenzymes were built by using electrostatic interaction, metal coordination, and host-guest interaction. The unique redox properties of selenoenzyme glutathione peroxidase (GPx) can be reproduced.

Computational Design and Study of Artificial Selenoenzyme with Controllable Activity Based on an Allosteric Protein Scaffold.

The establishment of new enzymatic function in an existing scaffold is a great challenge for protein engineers. In previous work, a highly efficient artificial selenoenzyme with controllable activity was constructed, based on a Ca2+ -responsive recoverin (Rn) protein. In this study, a design strategy combining docking, molecular dynamics, and MM-PBSA is presented, to predict the catalytically active site of glutathione peroxidase (GPx) on the allosteric domain of Rn. The energy contributions of the binding hot spot residues are evaluated further by energy decomposition analysis to determine the detailed substrate recognition mechanism of Rn, which provides clear guidance for artificial enzyme design for improved substrate binding (Michaelis-Menten constant, Km ).

Cover Feature: Computational Design and Study of Artificial Selenoenzyme with Controllable Activity Based on an Allosteric Protein Scaffold (Chem. Eur. J. 44/2019)

A computational protocol that combines docking, molecular dynamics, and MM-PBSA was described to guide the design of an artificial selenoenzyme based on a Ca2+-responsive recoverin (Rn) protein. The catalytically active site of glutathione peroxidase (GPx) can be precisely incorporated into the allosteric domain of Rn, leading to a high GPx activity and tightly controlled function for the catalyzed reduction of H2O2 by glutathione (GSH). These results provide important insights into how to create an enzyme-like site in non-homologous protein scaffolds. More information can be found in the Full Paper by Q. Luo et al. on page 10350.

Reversible pH-controlled switching of an artificial antioxidant selenoenzyme based on pseudorotaxane formation and dissociation.

A pH-responsive artificial selenoenzyme was constructed by reversible binding between organoselenium compound a1 nd CB[6] to form a pseudorotaxane-based molecular switch in response to pH stimuli. The glutathione peroxidase (GPx) activity of the artificial selenoenzyme can be switched on/off in a mild and body suitable environment between pH = 7 and pH = 6.

Reversible Ca2+ Switch of An Engineered Allosteric Antioxidant Selenoenzyme

AbstractA Ca2+‐responsive artificial selenoenzyme was constructed by computational design and engineering of recoverin with the active center of glutathione peroxidase (GPx). By combining the recognition capacity for the glutathione (GSH) substrate and the steric orientation of the catalytic selenium moiety, the engineered selenium‐containing recoverin exhibits high GPx activity for the catalyzed reduction of H2O2 by glutathione (GSH). Moreover, the engineered selenoenzyme can be switched on/off by Ca2+‐induced allosterism of the protein recoverin. This artificial selenoenzyme also displays excellent antioxidant ability when it was evaluated using a mitochondrial oxidative damage model, showing great potential for controlled catalysis in biomedical applications.

Reversible Ca(2+) switch of an engineered allosteric antioxidant selenoenzyme.

A Ca(2+) -responsive artificial selenoenzyme was constructed by computational design and engineering of recoverin with the active center of glutathione peroxidase (GPx). By combining the recognition capacity for the glutathione (GSH) substrate and the steric orientation of the catalytic selenium moiety, the engineered selenium-containing recoverin exhibits high GPx activity for the catalyzed reduction of H2 O2 by glutathione (GSH). Moreover, the engineered selenoenzyme can be switched on/off by Ca(2+) -induced allosterism of the protein recoverin. This artificial selenoenzyme also displays excellent antioxidant ability when it was evaluated using a mitochondrial oxidative damage model, showing great potential for controlled catalysis in biomedical applications.

Construction of a highly stable artificial glutathione peroxidase on a protein nanoring

Stable Protein One (SP1) is a boiling-stable oligomeric protein. The unique characteristics of SP1 offer a scaffold to design artificial enzymes against extreme temperature. Here, an efficient antioxidase is successfully constructed on the ring-shaped SP1 homododecamer. By means of computational design and genetic engineering, the active center of glutathione peroxidase (GPx), selenocysteine (Sec), is introduced to the SP1 monomer surface, and the self-assembly properties of the protein monomer lead to a ring-shaped SP1 with homododecamer catalytic selenium centers. This artificial selenoenzyme exhibits high GPx catalytic activity and shows a typical ping-pong kinetic mechanism. Moreover, it has a significantly broader temperature range and high thermostability. Owing to having multi-GPx active centers on a SP1 oligomer, this selenium-containing biomacromolecule exerts an excellent capability to protect cells from oxidative damage at the mitochondrial level. This strategy represents a new way to develop thermostable artificial nanoenzymes for some specific applications.

Engineering human seleno-glutaredoxin containing consecutive rare codons as an artificial glutathione peroxidase

The active center of human glutaredoxin (hGrx1) shares a common thioredoxin fold and specific affinity for substrate glutathione (GSH) with natural glutathione peroxidase (GPx). hGrx1 was redesigned to introduce the catalytic selenocysteine residue to imitate the function of antioxidant selenoenzyme GPx in vivo. The human hGrx1 scaffold is a good candidate for potential medical application compared with other animal-originated protein scaffolds. Two consecutive rare codons (AGG-AGG) in the open reading frame of hGrx1 mRNA encoding Arg26–Arg27 residues can reduce seleno-hGrx1 expression level significantly in the Cys auxotrophic Escherichia coli strain BL21cysE51. Therefore, we optimized the rare codons, which resulted in a remarkable increase of the expression level in the Cys auxotrophic cells, which may be sufficient for future medical production. The engineered artificial selenoenzyme displays high GPx catalytic activity, rivaling that of some natural GPx proteins. Kinetic analysis of the engineered seleno-hGrx1 showed a typical ping-pong kinetic mechanism; its catalytic properties are similar to those of some naturally occurring GPx proteins.

ChemInform Abstract: Artificial Selenoenzymes: Designed and Redesigned

AbstractReview: ca. 80 refs.

Artificial selenoenzymes: Designed and redesigned

Enzymes, highly evolved machinery developed by nature, catalyse reactions with formidable efficiency and specificity under mild conditions. Considerable efforts have been devoted for several decades on the development of enzyme-like catalysts with tailored properties by rationally manipulating natural and artificially synthesized host molecules. One of the great challenges is to design artificial systems with catalytic efficiencies and specificities rivalling natural components. Although most of the designed artificial enzymes present mild rate promotion, the high efficiency and specificity rivalling natural ones by artificially designed system appears. In this tutorial review, we recount the methods and strategies of design and redesign of artificial selenoenzymes on synthesized and natural hosts, with emphasis on construction of the active sites of antioxidative glutathione peroxidase (GPx) by the concept of synergy between recognition and catalysis (66 references).

New strategies for the design of artificial selenoenzymes

As one of the antioxidative enzymes exerting important biological roles in living bodies, the selenoenzyme glutathione peroxidase has received increased attentions. Over the past decades, much effort has been paid to mimic the catalytic behavior of glutathione peroxidase for exploring the enzymatic catalytic mechanisms and for developing potential antioxidative medicines. A great number of artificial GPxs, ranging from small molecular GPx mimics to macromolecular ones, and to nanoenzymes have been designed and prepared based on chemical, biological and supramolecular strategies. In this article, we specify the ideas and strategies for developing artificial GPxs based on macromolecules as scaffolds. The artificial system contains synthesized macromolecule-based selenoenzymes, self-assembled selenoenzymes, biomacromolecule-based selenoenzymes and so on.

Design of Artificial Selenoenzymes Based on Macromolecular Scaffolds

The selenoenzyme glutathione peroxidase has received increased attention as one of the antioxidative enzymes exerting important biological roles in living bodies. Over the past decades, much effort has been invested to mimic its catalytic behavior for understanding enzymatic catalytic mechanisms and also for developing potential medicines. A great number of artificial GPxs, ranging from small molecular compounds to macromolecular ones, have been designed and prepared by combining the concept of recognition and catalysis using chemical, biological and supramolecular strategies. In this article, we specify the development of artificial GPxs based on macromolecules as scaffolds, and discuss the power of reduced models in studying the bio-catalytic nature of selenoenzymes.

Selenoglutaredoxin as a Glutathione Peroxidase Mimic

Glutaredoxin (Grx1) from Escherichia coli is a monomeric, 85-amino-acid-long, disulfide-containing redox protein. A Grx1 variant in which the redox-active disulfide was replaced with a selenocysteine (C11U/C14S) was prepared by native chemical ligation from three fragments as a potential mimic of the natural selenoenzyme glutathione peroxidase (Gpx). Selenoglutaredoxin, like the analogous C14S Grx1 variant, shows weak peroxidase activity. The selenol provides a 30-fold advantage over the thiol, but its activity is four orders of magnitude lower than that of bovine Gpx. In contrast, selenoglutaredoxin is an excellent catalyst for thiol-disulfide exchange reactions; it promotes the reduction of beta-hydroxyethyldisulfide by glutathione with a specific activity of 130 units mg(-1). This value is 1.8 times greater than that of C14S Grx1 under identical conditions, and >10(4) greater than the peroxidase activity of either enzyme. Given the facile reduction of the glutathionyl-selenoglutaredoxin adduct by glutathione, oxidation of the selenol by the alkyl hydroperoxide substrate likely limits catalytic turnover and will have to be optimized to create more effective Gpx mimics. These results highlight the challenge of generating Gpx activity in a small, generic protein scaffold, despite the presence of a well-defined glutathione binding site and the intrinsic advantage of selenium over sulfur derivatives.

Selenocysteine.

Selenocysteine is recognized as the 21st amino acid in ribosome-mediated protein synthesis and its specific incorporation is directed by the UGA codon. Unique tRNAs that have complementary UCA anticodons are aminoacylated with serine, the seryl-tRNA is converted to selenocysteyl-tRNA and the latter binds specifically to a special elongation factor and is delivered to the ribosome. Recognition elements within the mRNAs are essential for translation of UGA as selenocysteine. A reactive oxygen-labile compound, selenophosphate, is the selenium donor required for synthesis of selenocysteyl-tRNA. Selenophosphate synthetase, which forms selenophosphate from selenide and ATP, is found in various prokaryotes, eukaryotes, and archaebacteria. The distribution and properties of selenocysteine-containing enzymes and proteins that have been discovered to date are discussed. Artificial selenoenzymes such as selenosubtilisin have been produced by chemical modification. Genetic engineering techniques also have been used to replace cysteine residues in proteins with selenocysteine. The mechanistic roles of selenocysteine residues in the glutathione peroxidase family of enzymes, the 5' deiodinases, formate dehydrogenases, glycine reductase, and a few hydrogenases are discussed. In some cases a marked decrease in catalytic activity of an enzyme is observed when a selenocysteine residue is replaced with cysteine. This substitution caused complete loss of glycine reductase selenoprotein A activity.

Selenocysteine

Selenocysteine is recognized as the 21st amino acid in ribosome-mediated protein synthesis and its specific incorporation is directed by the UGA codon. Unique tRNAs that have complementary UCA anticodons are aminoacylated with serine, the seryl-tRNA is converted to selenocysteyl-tRNA and the latter binds specifically to a special elongation factor and is delivered to the ribosome. Recognition elements within the mRNAs are essential for translation of UGA as selenocysteine. A reactive oxygen-labile compound, selenophosphate, is the selenium donor required for synthesis of selenocysteyl-tRNA. Selenophosphate synthetase, which forms selenophosphate from selenide and ATP, is found in various prokaryotes, eukaryotes, and archaebacteria. The distribution and properties of selenocysteine-containing enzymes and proteins that have been discovered to date are discussed. Artificial selenoenzymes such as selenosubtilisin have been produced by chemical modification. Genetic engineering techniques also have been used to replace cysteine residues in proteins with selenocysteine. The mechanistic roles of selenocysteine residues in the glutathione peroxidase family of enzymes, the 5′ deiodinases, formate dehydrogenases, glycine reductase, and a few hydrogenases are discussed. In some cases a marked decrease in catalytic activity of an enzyme is observed when a selenocysteine residue is replaced with cysteine. This substitution caused complete loss of glycine reductase selenoprotein A activity.

Crystal structure of selenosubtilisin at 2.0-.ANG. resolution

The three-dimensional structure of selenosubtilisin, an artificial selenoenzyme, has been solved at 2.0-A resolution by the method of molecular replacement. Selenosubtilisin is a chemical derivative of the bacterial serine protease subtilisin in which the catalytically essential serine residue has been replaced with a selenocysteine. Its unique hydrolytic and redox properties reflect the intrinsic chemical reactivity of the selenium prosthetic group. Structural analysis of the modified protein reveals that the selenium moiety is selectively incorporated into the side chain of residue 221 and confirms the seleninic acid oxidation state expected from treatment of the enzyme with hydrogen peroxide prior to crystallization. Although the seleninic acid replaces the essential nucleophile in the enzyme's catalytic triad and introduces a negative charge into the active site, the interaction between His64 and Asp32 is not altered by the modification. Hydrogen bonds from the oxygen atoms of the seleninic acid to His64 and to Asn155 in the oxyanion hole confine the prosthetic group to a single well-defined conformation within the active site. These interactions thus provide a structural basis for understanding the seleninic acid's unusually low pKa, the enzyme's relatively sluggish rate of reaction with thiols, and its much more efficient peroxidase activity. Aside from the active site region, the structure of the protein is essentially the same as that previously reported for native subtilisin Carlsberg, indicating the viability of chemical modification strategies for incorporating site-specific changes into the protein backbone. Comparison of the three-dimensional structures of selenosubtilisin and glutathione peroxidase, an important naturally occurring selenoenzyme, provides the means to evaluate how the function of the selenium prosthetic group varies with molecular context.