Dynamic evolution of selenocysteine utilization in bacteria: a balance between selenoprotein loss and evolution of selenocysteine from redox active cysteine residues

BACKGROUND: Selenocysteine (Sec) is co-translationally inserted into protein in response to UGA codons. It occurs in oxidoreductase active sites and often is catalytically superior to cysteine (Cys). However, Sec is used very selectively in proteins and organisms. The wide distribution of Sec and its restricted use have not been explained. RESULTS: We conducted comparative genomics and phylogenetic analyses to examine dynamics of Sec decoding in bacteria at both selenium utilization trait and selenoproteome levels. These searches revealed that 21.5% of sequenced bacteria utilize Sec, their selenoproteomes have 1 to 31 selenoproteins, and selenoprotein-rich organisms are mostly Deltaproteobacteria or Firmicutes/Clostridia. Evolutionary histories of selenoproteins suggest that Cys-to-Sec replacement is a general trend for most selenoproteins. In contrast, only a small number of Sec-to-Cys replacements were detected, and these were mostly restricted to formate dehydrogenase and selenophosphate synthetase families. In addition, specific selenoprotein gene losses were observed in many sister genomes. Thus, the Sec/Cys replacements were mostly unidirectional, and increased utilization of Sec by existing protein families was counterbalanced by loss of selenoprotein genes or entire selenoproteomes. Lateral transfers of the Sec trait were an additional factor, and we describe the first example of selenoprotein gene transfer between archaea and bacteria. Finally, oxygen requirement and optimal growth temperature were identified as environmental factors that correlate with changes in Sec utilization. CONCLUSION: Our data reveal a dynamic balance between selenoprotein origin and loss, and may account for the discrepancy between catalytic advantages provided by Sec and the observed low number of selenoprotein families and Sec-utilizing organisms

Identification of Trace Element-Containing Proteins in Genomic Databases

Development of bioinformatics tools provided researchers with the ability to identify full sets of trace element–containing proteins in organisms for which complete genomic sequences are available. Recently, independent bioinformatics methods were used to identify all, or almost all, genes encoding selenocysteine-containing proteins in human, mouse, and Drosophila genomes, characterizing entire selenoproteomes in these organisms. It also should be possible to search for entire sets of other trace element–associated proteins, such as metal-containing proteins, although methods for their identification are still in development

Adjustments, extinction, and remains of selenocysteine incorporation machinery in the nematode lineage

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date. After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International).Selenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Secincorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model.This work was supported by Universidad de la República, Uruguay (Grant Number 418 to G.S., PhD fellowship to L.O.); Asociación Española de Cooperación Internacional (C/7646/07 to A.M.-V. and G.S.; A/016083/08 to A.M.-V. and G.S.); Asociación Universitaria Iberoamericana de Posgrado and Agencia Nacional de Innovación e Investigación (BE_POS_2009_183 and BE_POS_2010_2160 to L.O.), and was partially funded by FOCEM (MERCOSUR Structural Convergence Fund), [COF 03/11].Peer Reviewe

Evolution of selenium utilization traits

BACKGROUND: The essential trace element selenium is used in a wide variety of biological processes. Selenocysteine (Sec), the 21st amino acid, is co-translationally incorporated into a restricted set of proteins. It is encoded by an UGA codon with the help of tRNA(Sec )(SelC), Sec-specific elongation factor (SelB) and a cis-acting mRNA structure (SECIS element). In addition, Sec synthase (SelA) and selenophosphate synthetase (SelD) are involved in the biosynthesis of Sec on the tRNA(Sec). Selenium is also found in the form of 2-selenouridine, a modified base present in the wobble position of certain tRNAs, whose synthesis is catalyzed by YbbB using selenophosphate as a precursor. RESULTS: We analyzed completely sequenced genomes for occurrence of the selA, B, C, D and ybbB genes. We found that selB and selC are gene signatures for the Sec-decoding trait. However, selD is also present in organisms that do not utilize Sec, and shows association with either selA, B, C and/or ybbB. Thus, selD defines the overall selenium utilization. A global species map of Sec-decoding and 2-selenouridine synthesis traits is provided based on the presence/absence pattern of selenium-utilization genes. The phylogenies of these genes were inferred and compared to organismal phylogenies, which identified horizontal gene transfer (HGT) events involving both traits. CONCLUSION: These results provide evidence for the ancient origin of these traits, their independent maintenance, and a highly dynamic evolutionary process that can be explained as the result of speciation, differential gene loss and HGT. The latter demonstrated that the loss of these traits is not irreversible as previously thought

RNA-dependent selenocysteine biosynthesis in eukaryotes and Archaea

Selenocysteine (Sec), the 21st genetically encoded amino acid, is the major metabolite of the micronutrient selenium. Sec is inserted into nascent proteins in response to a UGA codon. The substrate for ribosomal protein synthesis is selenocysteinyl-tRNASec. While the formation of Sec-tRNASec from seryl-tRNASec by a single bacterial enzyme selenocysteine synthase (SelA) has been well described, the mechanism of Sec-tRNASec formation in archaea and eukaryotes remained poorly understood. Herein, biochemical and genetic data provide evidence that, in contrast to bacteria, eukaryotes and archaea utilize a different route to Sec-tRNASec that requires the tRNASec-dependent conversion of O-phosphoserine (Sep) to Sec. In this two-step pathway, O-phosphoseryl-tRNA kinase (PSTK) first converts Ser-tRNASec to Sep-tRNASec. This misacylated tRNA is the obligatory precursor for a Sep-tRNA: Sec-tRNA synthase (SepSecS); this protein was previously annotated as Soluble Liver Antigen/Liver Pancreas (SLA/LP). SepSecS genes from Homo sapiens, the lower eukaryote Trypanosoma brucei and the archaea Methanocaldococcus jannaschii and Methanococcus maripaludis complement an Escherichia coli DeltaselA deletion strain in vivo. Furthermore, genetic analysis of selenoprotein biosynthesis in T. brucei in vivo demonstrated that eukaryotes have a single pathway to Sec-tRNASec that requires Sep-tRNASec as an intermediate. Finally, purified recombinant SepSecS converts Sep-tRNA Sec into Sec-tRNASec in vitro in the presence of sodium selenite and purified E. coli selenophosphate synthetase.The final step in Sec biosynthesis was further investigated by a structure-based mutational analysis of the M. maripaludis SepSecS and by determining the crystal structure of human SepSecS complexed with tRNA Sec, phosphoserine and thiophosphate at 2.8 A resolution. In vivo and in vitro enzyme assays support a mechanism of Sec-tRNASec formation based on pyridoxal phosphate, while the lack of active site cysteines demonstrates that a perselenide intermediate is not involved in SepSecS-catalyzed Sec formation. Two tRNASec molecules, with a fold distinct from other canonical tRNAs, bind to each human SepSecS tetramer through their unique 13 base-pair acceptor-TPsiC arm. The tRNA binding induces a conformational change in the enzyme\u27s active site that allows a Sep covalently attached to tRNASec, but not free Sep, to be oriented properly for the reaction to occur

A computational method to predict genetically encoded rare amino acids in proteins

In several natural settings, the standard genetic code is expanded to incorporate two additional amino acids with distinct functionality, selenocysteine and pyrrolysine. These rare amino acids can be overlooked inadvertently, however, as they arise by recoding at certain stop codons. We report a method for such recoding prediction from genomic data, using read-through similarity evaluation. A survey across a set of microbial genomes identifies almost all the known cases as well as a number of novel candidate proteins

A Novel Protein Kinase-Like Domain in a Selenoprotein, Widespread in the Tree of Life

Selenoproteins serve important functions in many organisms, usually providing essential oxidoreductase enzymatic activity, often for defense against toxic xenobiotic substances. Most eukaryotic genomes possess a small number of these proteins, usually not more than 20. Selenoproteins belong to various structural classes, often related to oxidoreductase function, yet a few of them are completely uncharacterised

Evolution of selenophosphate synthetases: emergence and relocation of function through independent duplications and recurrent subfunctionalization

Selenoproteins are proteins that incorporate selenocysteine (Sec), a nonstandard amino acid encoded by UGA, normally a stop codon. Sec synthesis requires the enzyme Selenophosphate synthetase (SPS or SelD), conserved in all prokaryotic and eukaryotic genomes encoding selenoproteins. Here, we study the evolutionary history of SPS genes, providing a map of selenoprotein function spanning the whole tree of life. SPS is itself a selenoprotein in many species, although functionally equivalent homologs that replace the Sec site with cysteine (Cys) are common. Many metazoans, however, possess SPS genes with substitutions other than Sec or Cys (collectively referred to as SPS1). Using complementation assays in fly mutants, we show that these genes share a common function, which appears to be distinct from the synthesis of selenophosphate carried out by the Sec- and Cys- SPS genes (termed SPS2), and unrelated to Sec synthesis. We show here that SPS1 genes originated through a number of independent gene duplications from an ancestral metazoan selenoprotein SPS2 gene that most likely already carried the SPS1 function. Thus, in SPS genes, parallel duplications and subsequent convergent subfunctionalization have resulted in the segregation to different loci of functions initially carried by a single gene. This evolutionary history constitutes a remarkable example of emergence and evolution of gene function, which we have been able to trace thanks to the singular features of SPS genes, wherein the amino acid at a single site determines unequivocally protein function and is intertwined to the evolutionary fate of the entire selenoproteome

The Physiology and Evolution of Selenite Respiration in Bacteria

The selenium oxyanions selenate (Se(VI)) and selenite (Se(IV)) can be utilized by some bacteria and archaea as terminal electron acceptors in anaerobic respiration. Se(VI) and Se(IV) respiration is mediated by a phylogenetically and ecologically diverse array of organisms, suggesting that selenium respiration is ubiquitous in natural environments. Several respiratory Se(VI) reductases have been characterized in bacteria, revealing that Se(VI) respiration has evolved independently several times in this domain. Se(IV) respiration, in contrast, has yet to be characterized. I have purified and characterized the first respiratory Se(IV) reductase from Bacillus selenitireducens MLS10. The Se(IV) reductase appeared to purify as a single 80 kDa enzyme and contained both iron and molybdenum. The enzyme was highly specific for Se(IV). The genome of MLS10 demonstrated that this enzyme was part of an operon encoding a putative respiratory Se(IV) reductase (Srr) complex. Srr was electrophoretically purified from MLS10 periplasm using non-denaturing gels, and identified using in-gel enzyme assays for Se(IV) reducing activity. Multiple subunits from Srr were identified using liquid chromatography tandem mass spectrometry, confirming the operon codes for a Srr complex. The enzyme was designated SrrA. Phylogenetic analysis determined that SrrA was a member of the polysulfide reductase catalytic subunit (PsrA) and thiosulfate reductase catalytic subunit (PhsA) lineage of molybdopterin or tungstopterin bis(pyranopterin guanine dinucleotide) (Mo/W-bisPGD)-containing molybdoenzymes. Analysis of the operons associated with each catalytic subunit in the phylogeny revealed that putative PsrA, PhsA, and SrrA homologs did not form monophyletic clades with respect to one another. Furthermore, while putative Srr operons were not observed in archaea, Srr was present throughout the PsrA/PhsA lineage in bacteria. To determine if Srr is a reliable marker for Se(IV) respiration, I attempted to ascertain if two organisms, Bacillus beveridgei MLTeJB and Desulfitobacterium hafniense PCP-1, expressed Srr when cultivated in the presence of Se(IV). MLTeJB was shown to express SrrA when grown on both Se(VI) and Se(IV). Definitive identification of the Se(IV) reductase expressed by PCP-1 when grown on Se(VI) was not possible due to the low expression levels of the enzyme. This work represents the first physiological and evolutionary studies of Se(IV) respiration

A short motif in Drosophila SECIS Binding Protein 2 provides differential binding affinity to SECIS RNA hairpins

Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding UGA Sec requires a complex mechanism, comprising the cis-acting SECIS RNA hairpin in the 3′UTR of selenoprotein mRNAs, and trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central to the mechanism. SBP2 has been so far functionally characterized only in rats and humans. In this work, we report the characterization of the Drosophila melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same selenoprotein synthesis-promoting capabilities as the mammalian counterpart. However, a major difference resides in the SECIS recognition pattern: while human SBP2 (hSBP2) binds the distinct form 1 and 2 SECIS RNAs with similar affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we report the identification of a K (lysine)-rich domain in all SBP2s, essential for SECIS and 60S ribosomal subunit binding, differing from the well-characterized L7Ae RNA-binding domain. Swapping only five amino acids between dSBP2 and hSBP2 in the K-rich domain conferred reversed SECIS-binding properties to the proteins, thus unveiling an important sequence for form 1 binding