A 4-selenocysteine, 2-selenocysteine insertion sequence (SECIS) element methionine sulfoxide reductase from Metridium senile reveals a non-catalytic function of selenocysteines

Selenocysteine (Sec) residues occur in thiol oxidoreductase families, and functionally characterized selenoenzymes typically have a single Sec residue used directly for redox catalysis. However, how new Sec residues evolve and whether non-catalytic Sec residues exist in proteins is not known. Here, we computationally identified several genes with multiple Sec insertion sequence (SECIS) elements, one of which was a methionine-R-sulfoxide reductase (MsrB) homolog from Metridium senile that has four in-frame UGA codons and two nearly identical SECIS elements. One of the UGA codons corresponded to the conserved catalytic Sec or Cys in MsrBs, whereas the three other UGA codons evolved recently and had no homologs with Sec or Cys in these positions. Metabolic (75)Se labeling showed that all four in-frame UGA codons supported Sec insertion and that both SECIS elements were functional and collaborated in Sec insertion at each UGA codon. Interestingly, recombinant M. senile MsrB bound iron, and further analyses suggested the possibility of binding an iron-sulfur cluster by the protein. These data show that Sec residues may appear transiently in genes containing SECIS elements and be adapted for non-catalytic functions.     

Engineered selenium-containing glutaredoxin displays strong glutathione peroxidase activity rivaling natural enzyme

Insertion of selenocysteine (Sec) into protein scaffolds provides an opportunity for designing enzymes with improved and unusual catalytic properties. The use of a common thioredoxin fold with a high affinity for glutathione in glutaredoxin (Grx) and glutathione peroxidase (GPx) suggests a possibility of engineering Grx into GPx and vice versa. Here, we engineered a Grx domain of mouse thioredoxin/glutathione reductase (TGR) into a selenium-containing enzyme by substituting the active site cysteine (Cys) with selenocysteine (Sec) in a Cys auxotrophic system. The resulting selenoenzyme displayed an unusually high GPx catalytic activity rivaling that of several native GPxs. The engineered seleno-Grx was characterized by mass spectrometry and kinetic analyses. It showed a typical ping-pong kinetic mechanism, and its catalytic properties were similar to those of naturally occurring GPxs. For example, its second rate constant (k(cat)/K(mH2O2)) was as high as 1.55x10(7) M(-1) min(-1). It appears that glutathione-dependent Grx, GPx and glutathione transferase (GST) evolved from a common thioredoxin-like ancestor to accommodate related glutathione-dependent functions and can be interconverted by targeted Sec insertion.     

Purification and properties of a recombinant sulfur analog of murine selenium-glutathione peroxidase.

We previously constructed plasmids for synthesis of glutathione-peroxidase (GPx) mutants in an Escherichia coli expression system. In these recombinant proteins either cysteine ([Cys]GPx mutant) or serine ([Ser]GPx mutant) were present in place of the active-site selenocysteine (SeCys) of the natural enzyme. We have now investigated GPx activity of [Cys]GPx and [Ser]GPx mutants. Enzyme assays performed on preparations of these partially purified proteins demonstrated that the [Cys]GPx mutant exhibited a significant GPx activity, unlike the [Ser]GPx mutant. Purification of [Cys]GPx was performed in two steps of ion-exchange chromatography giving a 98% homogenous protein in 50% yield. The purified [Cys]GPx protein was shown to be a symmetrical tetramer by the means of gel-filtration HPLC and SDS/PAGE. Two isoelectric points were found (6.8 and 7.2) which may reflect two different oxidation states of the mutant protein. The GPx activity of the [Cys]GPx mutant was optimal at pH 8.5. The [Cys]GPx mutant had a specific activity approximately 1000-fold smaller than that of the natural enzyme, and was very easily inactivated by hydroperoxides. Inhibition of the activity with iodoacetate determined a pKa of 8.3, presumably that of the active-site cysteine. Unlike that of SeGPx, the GPx activity of [Cys]GPx was only slightly inhibited by mercaptosuccinate. We discuss hypothetical mechanistic constraints of either catalytic cycle, which may explain such results.     

The selenoproteome of Clostridium sp. OhILAs: characterization of anaerobic bacterial selenoprotein methionine sulfoxide reductase A

Selenocysteine (Sec) is incorporated into proteins in response to UGA codons. This residue is frequently found at the catalytic sites of oxidoreductases. In this study, we characterized the selenoproteome of an anaerobic bacterium, Clostridium sp. (also known as Alkaliphilus oremlandii) OhILA, and identified 13 selenoprotein genes, five of which have not been previously described. One of the detected selenoproteins was methionine sulfoxide reductase A (MsrA), an antioxidant enzyme that repairs oxidatively damaged methionines in a stereospecific manner. To date, little is known about MsrA from anaerobes. We characterized this selenoprotein MsrA which had a single Sec residue at the catalytic site but no cysteine (Cys) residues in the protein sequence. Its SECIS (Sec insertion sequence) element did not resemble those in Escherichia coli. Although with low translational efficiency, the expression of the Clostridium selenoprotein msrA gene in E. coli could be demonstrated by (75)Se metabolic labeling, immunoblot analyses, and enzyme assays, indicating that its SECIS element was recognized by the E. coli Sec insertion machinery. We found that the Sec-containing MsrA exhibited at least a 20-fold higher activity than its Cys mutant form, indicating a critical role of Sec in the catalytic activity of the enzyme. Furthermore, our data revealed that the Clostridium MsrA was inefficiently reducible by thioredoxin, which is a typical reducing agent for MsrA, suggesting the use of alternative electron donors in this anaerobic bacterium that directly act on the selenenic acid intermediate and do not require resolving Cys residues.     

Different Catalytic Mechanisms in Mammalian Selenocysteine- and Cysteine-Containing Methionine-R-Sulfoxide Reductases

Selenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are uniquely conserved in Cys-containing MsrBs and which are critical for enzyme activity in MsrB2 and MsrB3, were not required for MsrB1, but increased the activity of its Cys mutant. Conversely, selenoprotein MsrB1 had a unique resolving Cys reversibly engaged in the selenenylsulfide bond. However, this Cys was not necessary for activities of either MsrB2, MsrB3, or the Cys mutant of MsrB1. We prepared Sec-containing forms of MsrB2 and MsrB3 and found that they were more than 100-fold more active than the natural Cys forms. However, these selenoproteins could not be reduced by the physiological electron donor, thioredoxin. Yet, insertion of the resolving Cys, which was conserved in MsrB1, into the selenoprotein form of MsrB3 restored the thioredoxin-dependent activity of this enzyme. These data revealed differences in catalytic mechanisms between selenoprotein MsrB1 and non-selenoproteins MsrB2 and MsrB3, and identified catalytic advantages and disadvantages of Sec- and Cys-containing proteins. The data also suggested that Sec- and Cys-containing oxidoreductases require distinct sets of active-site features that maximize their catalytic efficiencies and provide strategies for protein design with improved catalytic properties.     

Different catalytic mechanisms in mammalian selenocysteine- and cysteine-containing methionine-R-sulfoxide reductases

Selenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are uniquely conserved in Cys-containing MsrBs and which are critical for enzyme activity in MsrB2 and MsrB3, were not required for MsrB1, but increased the activity of its Cys mutant. Conversely, selenoprotein MsrB1 had a unique resolving Cys reversibly engaged in the selenenylsulfide bond. However, this Cys was not necessary for activities of either MsrB2, MsrB3, or the Cys mutant of MsrB1. We prepared Sec-containing forms of MsrB2 and MsrB3 and found that they were more than 100-fold more active than the natural Cys forms. However, these selenoproteins could not be reduced by the physiological electron donor, thioredoxin. Yet, insertion of the resolving Cys, which was conserved in MsrB1, into the selenoprotein form of MsrB3 restored the thioredoxin-dependent activity of this enzyme. These data revealed differences in catalytic mechanisms between selenoprotein MsrB1 and non-selenoproteins MsrB2 and MsrB3, and identified catalytic advantages and disadvantages of Sec- and Cys-containing proteins. The data also suggested that Sec- and Cys-containing oxidoreductases require distinct sets of active-site features that maximize their catalytic efficiencies and provide strategies for protein design with improved catalytic properties.     

Substituting selenocysteine for catalytic cysteine 41 enhances enzymatic activity of plant phospholipid hydroperoxide glutathione peroxidase expressed in Escherichia coli

The citrus phospholipid hydroperoxide glutathione peroxidase (cit-PHGPx) was the first plant peroxidase demonstrated to exhibit PHGPx-specific enzymatic activity, although it was 500-fold weaker than that of the pig heart analog. This relatively low activity is accounted for the catalytic residue of cit-PHGPx, which was found to be cysteine and not the rare selenocysteine (Sec) present in animal enzymes. Sec incorporation into proteins is encoded by a UGA codon, usually a STOP codon, which, in prokaryotes, is suppressed by an adjacent downstream mRNA stem-loop structure, the Sec insertion sequence (SECIS). By performing appropriate nucleotide substitutions into the gene encoding cit-PHGPx, we introduced bacterial-type SECIS elements that afforded the substitution of the catalytic Cys(41) by Sec, as established by mass spectrometry, while preserving the functional integrity of the peroxidase. The recombinant enzyme, whose synthesis is selenium-dependent, displayed a 4-fold enhanced peroxidase activity as compared with the Cys-containing analog, thus confirming the higher catalytic power of Sec compared with Cys in cit-PHGPx active site. The study led also to refinement of the minimal sequence requirements of the bacterial-type SECIS, and, for the first time, to the heterologous expression in Escherichia coli of a eukaryotic selenoprotein containing a SECIS in its open reading frame.     

Selenocysteine in proteins—properties and biotechnological use

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK_a of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of ⁷⁷Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.     

A selenocysteine-containing peroxiredoxin from the strictly anaerobic organism Eubacterium acidaminophilum

A strongly 75Se-labeled 22 kDa protein detected previously showed in its N-terminal sequence the highest similarity to the family of thiol-dependent peroxidases, now called peroxiredoxins. The respective gene prxU was cloned and analyzed. prxU encodes a protein of 203 amino acids (22,470 Da) and contains an in-frame UGA codon (selenocysteine) at the position of the so far strictly conserved and catalytically active Cys47. The second conserved cysteine present in 2-Cys peroxiredoxins was replaced by alanine. Heterologous expression of the Eubacterium acid-aminophilum PrxU as a recombinant selenoprotein in Escherichia coli was not possible. A cysteine-encoding mutant gene, prxU47C, containing UGC instead of UGA was strongly expressed. This recombinant PrxU47C mutant protein was purified to homogeneity by its affinity tag, but was not active as a thiol-dependent peroxidase. The identification of prxU reveals that the limited class of natural selenoproteins may in certain organisms also include isoenzymes of peroxiredoxins, previously only known as non-selenoproteins containing catalytic cysteine residues.     

Selective restoration of the selenoprotein population in a mouse hepatocyte selenoproteinless background with different mutant selenocysteine tRNAs lacking Um34

Novel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA([Ser]Sec) gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T-->A) or 37 (37A-->G) in tRNA([Ser]Sec). The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2'-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N(6)-isopentenyladenosine. Both mutant tRNAs lacked the 2'-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with (75)Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins.     

Selenocysteine in proteins-properties and biotechnological use

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK(a) of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.     

Cysteine is the presumed catalytic residue of Citrus sinensis phospholipid hydroperoxide glutathione peroxidase over‐expressed under salt stress

A citrus salt‐stress associated protein (Cit‐SAP), partially purified from citrus cultured cells, was previously identified as the first plant phospholipid hydroperoxide glutathione peroxidase (PHGPx). The nucleotide sequence of its isolated gene ( csa ) revealed that a TGT, known as codon for Cys, encodes the presumed catalytic residue 41 in the polypeptide chain of Cit‐SAP. In animals, a TGA encodes the rare amino acid selenocysteine (Sec) as the catalytic residue of the analogous enzyme. It is of interest to establish whether the TGT codon for this catalytic residue in the plant enzyme is indeed translated to Cys and not to Sec, and to demonstrate the effect of such a change, if it exists, on the nature of the enzymatic activity of the plant enzyme as compared to that of the animal. In the present study, we have purified for the first time, by affinity chromatography, enzymatically active citrus PHGPx from recombinant Escherichia coli bearing the csa gene. Tryptic digestion of the purified enzyme followed by HPLC afforded the isolation of a peptide which contains residue 41, and its sequence analysis revealed that this residue is indeed a Cys, and not Sec. The enzymatic activity and specificity of the recombinant Cit‐SAP was found to be similar to that observed before for the partially purified plant enzyme. However, the rate of this activity was much lower towards phospholipid hydroperoxides, and none towards hydrogen peroxide, as compared to that of the animal analogue. It is therefore suggested that the presence of a Cys, and not Sec, as the catalytic residue in the plant enzyme, affects its enzymatic activity and may determine a different biological role for the plant PHGPx from that of the animal.     

The thioredoxin specificity of Drosophila GPx: a paradigm for a peroxiredoxin-like mechanism of many glutathione peroxidases

Some members of the glutathione peroxidase (GPx) family have been reported to accept thioredoxin as reducing substrate. However, the selenocysteine-containing ones oxidise thioredoxin (Trx), if at all, at extremely slow rates. In contrast, the Cys homolog of Drosophila melanogaster exhibits a clear preference for Trx, the net forward rate constant, k'(+2), for reduction by Trx being 1.5x10(6) M(-1) s(-1), but only 5.4 M(-1) s(-1) for glutathione. Like other CysGPxs with thioredoxin peroxidase activity, Drosophila melanogaster (Dm)GPx oxidized by H(2)O(2) contained an intra-molecular disulfide bridge between the active-site cysteine (C45; C(P)) and C91. Site-directed mutagenesis of C91 in DmGPx abrogated Trx peroxidase activity, but increased the rate constant for glutathione by two orders of magnitude. In contrast, a replacement of C74 by Ser or Ala only marginally affected activity and specificity of DmGPx. Furthermore, LC-MS/MS analysis of oxidized DmGPx exposed to a reduced Trx C35S mutant yielded a dead-end intermediate containing a disulfide between Trx C32 and DmGPx C91. Thus, the catalytic mechanism of DmGPx, unlike that of selenocysteine (Sec)GPxs, involves formation of an internal disulfide that is pivotal to the interaction with Trx. Hereby C91, like the analogous second cysteine in 2-cysteine peroxiredoxins, adopts the role of a "resolving" cysteine (C(R)). Molecular modeling and homology considerations based on 450 GPxs suggest peculiar features to determine Trx specificity: (i) a non-aligned second Cys within the fourth helix that acts as C(R); (ii) deletions of the subunit interfaces typical of tetrameric GPxs leading to flexibility of the C(R)-containing loop. Based of these characteristics, most of the non-mammalian CysGPxs, in functional terms, are thioredoxin peroxidases.     

An anaerobic bacterial MsrB model reveals catalytic mechanisms, advantages, and disadvantages provided by selenocysteine and cysteine in reduction of methionine-R-sulfoxide

We verified and generalized the catalytic features that selenocysteine (Sec) and cysteine (Cys) contribute to the reduction of methionine-R-sulfoxide using an anaerobic bacterial MsrB from Clostridium sp. OhILA as a model protein. The Sec-containing Clostridium MsrB form exhibited 100-fold higher activity than its Cys-containing form, revealing that Sec provided the catalytic advantage of higher activity. However, a resolving Cys was required for the thioredoxin (Trx)-dependent recycling process of the Sec-containing form. Thus, Trx could reduce the selenenylsulfide bond, but its Trx-dependent recycling process was much less efficient compared to that for the disulfide bond in the Cys-containing form, demonstrating an obvious catalytic disadvantage. These data agreed well with our previous data on mammalian MsrBs, and therefore suggested that the catalytic mechanisms, as well as the catalytic advantages and disadvantages provided by the Sec and Cys residues, are most likely conserved from anaerobic bacteria to mammals. Taken together, we propose that the use of Sec in MsrB may depend on a balance between the catalytic advantage of higher activity and the disadvantage of a less efficient regeneration process provided by this residue.     

Multiple cysteine residues are implicated in Janus kinase 2-mediated catalysis

The redox regulation of Janus kinase 2 (JAK2) is poorly understood, and there are contradictory reports as to whether the enzyme's activity is inhibited or stimulated by oxidizing conditions in the cell. Here we demonstrate that multiple cysteine residues within the JAK2 catalytic domain may be crucial for enzymatic activity. The enzyme is catalytically inactive when oxidized; activity can be restored via reduction to the thiol state. A series of recombinant variants of JAK2 were overproduced using the baculoviral expression vector system. A truncated variant of JAK2, GST/(NDelta661)rJAK2, provided evidence that the amino-terminal autoinhibitory domain was not essential for direct redox regulation and that only nine cysteine residues were potentially involved. The effect of individually and combinatorially altering these nine cysteines was examined via cysteine-to-serine mutagenesis. This identified four cysteine residues in the catalytic domain (Cys866, Cys917, Cys1094, and Cys1105) that cooperatively maintain JAK2's catalytic competency. Our data are consistent with a direct mechanism for redox regulation of JAK2 via oxidation and reduction of critical cysteine residues.     

Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase

Mammalian thioredoxin reductase [EC 1.6.4.5], a homodimeric flavoprotein, has a marked similarity to glutathione reductase. The two cysteines in the N-terminal FAD domain (-Cys59-x-x-x-x-Cys64-) and histidine (His472) are conserved between them at corresponding positions, but the mammalian thioredoxin reductase contains a C-terminal extension of selenocysteine (Sec or U) at the penultimate position and a preceding cysteine (-Gly-Cys497-Sec498-Gly). Introduction of mutations into the cloned rat thioredoxin reductase gene revealed that residues Cys59, Cys64, His472, Cys497, and Sec498, as well as the sequence of Cys497 and Sec498 were essential for thioredoxin-reducing activity. To analyze the catalytic mechanism of the mammalian thioredoxin reductase, the wild-type, U498C, U498S, C59S, and C64S were overproduced in a baculovirus/insect cell system and purified. The wild-type thioredoxin reductase produced in this system, designated as WT, was found to lack the Sec residue and to terminate at Cys497. A Sec-containing thioredoxin reductase, which was purified from COS-1 cells transfected with the wild-type cDNA, was designated as SecWT and was used as an authentic enzyme. Among mutant enzymes, only U498C retained a slight thioredoxin-reducing activity at about three orders magnitude lower than SecWT. WT, U498C, and U498S showed some 5,5'-dithiobis(2-nitrobenzoic acid)-reducing activity and transhydrogenase activity, and C59S and C64S had substantially no such activities. These data and spectral analyses of these enzymes suggest that Cys59 and Cys64 at the N-terminus, in conjunction with His472, function as primary acceptors for electrons from NADPH via FAD, and that the electrons are then transferred to Cys497-Sec498 at the C-terminus for the reduction of oxidized thioredoxin in the mammalian thioredoxin reductase.     

Characterization of Mycobacterium tuberculosis diaminopimelic acid epimerase: paired cysteine residues are crucial for racemization

Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K(M) value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis.     

Intein-based Design Expands Diversity of Selenocysteine Reporters

The presence of selenocysteine in a protein confers many unique properties that make the production of recombinant selenoproteins desirable. Targeted incorporation of Sec into a protein of choice is possible by exploiting elongation factor Tu-dependent reassignment of UAG codons, a strategy that has been continuously improved by a variety of means. Improving selenoprotein yield by directed evolution requires selection and screening markers that are titratable, have a high dynamic range, enable high-throughput screening, and can discriminate against nonspecific UAG decoding. Current screening techniques are limited to a handful of reporters where a cysteine (Cys) or Sec residue normally affords activity. Unfortunately, these existing Cys/Sec-dependent reporters lack the dynamic range of more ubiquitous reporters or suffer from other limitations. Here we present a versatile strategy to adapt established reporters for specific Sec incorporation. Inteins are intervening polypeptides that splice themselves from the precursor protein in an autocatalytic splicing reaction. Using an intein that relies exclusively on Sec for splicing, we show that this intein cassette can be placed in-frame within selection and screening markers, affording reporter activity only upon successful intein splicing. Furthermore, because functional splicing can only occur when a catalytic Sec is present, the amount of synthesized reporter directly measures UAG-directed Sec incorporation. Importantly, we show that results obtained with intein-containing reporters are comparable to the Sec incorporation levels determined by mass spectrometry of isolated recombinant selenoproteins. This result validates the use of these intein-containing reporters to screen for evolved components of a translation system yielding increased selenoprotein amounts.     

Sulfhydryl groups responsible for extreme lability of human cholesterol 7alpha-hydroxylase identified by site-directed mutagenesis

Cholesterol 7alpha-hydroxylase (cholesterol-NADPH oxidoreductase, EC 1.14.13.17, 7alpha-hydroxylating) is known to have extremely sensitive sulfhydryl group(s). It is believed that a cysteine residue that has a sulfhydryl group plays an important role in the decrease of this enzyme activity. The amino acid sequences of cholesterol 7alpha-hydroxylase of five different mammalian species, human, rat, rabbit, hamster and mouse, revealed that these mammalian species contain eight cysteine residues that are well conserved. To identify which cysteine residues are responsible for the extremely high lability, we used the technique of the site-directed mutagenesis. Eight mutated genes of human cholesterol 7alpha-hydroxylase in which one codon for a cysteine residue was changed to that for alanine were prepared and expressed in COS-1 cells. The protein mass and enzyme activity of cholesterol 7alpha-hydroxylse obtained from these eight mutated genes were determined. While all mutated genes expressed the enzyme mass, two mutated genes did not express protein capable of catalyzing 7alpha-hydroxylation of cholesterol: in one mutant a codon for the 7th cysteine residue (Cys 444) was substituted to that for alanine and in the other mutant a codon for the 8th cysteine residue (Cys 476) was changed similarly. These results suggest that the 7th and 8th cysteine residues are important for expression of the enzyme activity. Based on the fact that Cys 444 exists in the heme binding region, Cys 476 was suggested to be responsible for enzyme lability.     

A model for Sec incorporation with the regions upstream of the UGA Sec codon to play a key role

For eukaryotic selenoprotein mRNAs, it has been proposed that the SECIS element in the 3'-UTR is required for recognition of UGA as a Sec codon. Some proteins which bind to SECIS (SBP) have been reported. However, it is not clear how the SECIS element in the 3'-UTR can mediate Sec insertion far at the in-frame UGA Sec codons. The idea that there must be a signal near the UGA Sec codon is still being considered. Therefore, we searched for a protein which binds to an RNA sequence surrounding the UGA Sec codon on human GPx mRNA. We found a protein, prepared from bovine brain microsomes, which strongly bound to the RNA fragment upstream of the UGA Sec codon but not to the RNA sequence downstream of the UGA codon. This protein also bound to the SECIS sequence in the 3'-UTR of human GPx, and this binding to SECIS was competed with the RNA fragment upstream of the UGA Sec codon. We also obtained the similar results with the RNA fragments of type I iodothyronine 5'-deiodinase (5'DI) mRNAs. Comparison of such RNA fragments with SECIS fragments revealed similarities in the region upstream of the in-frame UGA Sec codon of several Se-protein mRNAs. The study thus favors a novel model of Sec incorporation at the UGA Sec codon that involves the regions upstream of the UGA codon of mRNAs of mammalian selenoproteins. This model explains that the stem-loop structure covering the UGA codon is recognized by SBP and how the UGA Sec codon escapes from attack by eRF.