Initial Step of Selenite Reduction via Thioredoxin for Bacterial Selenoprotein Biosynthesis.

Atsuki Shimizu,N Tejo Prakash,Takuya Ogawa,Riku Aono,Satoru Hagita,Kaito Kiriyama,Masao Inoue,Kei Goto,Hisaaki Mihara,Tatsuo Kurihara,Ryuta Tobe,Yosuke Toyotake

doi:10.3390/ijms222010965

Abstract

Many organisms reductively assimilate selenite to synthesize selenoprotein. Although the thioredoxin system, consisting of thioredoxin 1 (TrxA) and thioredoxin reductase with NADPH, can reduce selenite and is considered to facilitate selenite assimilation, the detailed mechanism remains obscure. Here, we show that selenite was reduced by the thioredoxin system from Pseudomonas stutzeri only in the presence of the TrxA (PsTrxA), and this system was specific to selenite among the oxyanions examined. Mutational analysis revealed that Cys33 and Cys36 residues in PsTrxA are important for selenite reduction. Free thiol-labeling assays suggested that Cys33 is more reactive than Cys36. Mass spectrometry analysis suggested that PsTrxA reduces selenite via PsTrxA-SeO intermediate formation. Furthermore, an in vivo formate dehydrogenase activity assay in Escherichia coli with a gene disruption suggested that TrxA is important for selenoprotein biosynthesis. The introduction of PsTrxA complemented the effects of TrxA disruption in E. coli cells, only when PsTrxA contained Cys33 and Cys36. Based on these results, we proposed the early steps of the link between selenite and selenoprotein biosynthesis via the formation of TrxA–selenium complexes.

Highlights

Selenium is an essential trace element in many organisms [1,2,3]
Selenite can serve as a nutritional source of selenium for bacteria
Selenite is provided from another inorganic selenium source, selenate, by selenate reductases such as E

Summary

Introduction

Selenium is an essential trace element in many organisms [1,2,3]. Most of its important roles in cells are exerted as the 21st amino acid selenocysteine (Sec) [4], which is translationally incorporated into selenoproteins such as formate dehydrogenase (FDH), glycine reductase, and hydrogenase in bacteria as well as glutathione peroxidases, selenoproteinP, and thioredoxin reductase (TXNRD) in mammals [5,6]. Most of its important roles in cells are exerted as the 21st amino acid selenocysteine (Sec) [4], which is translationally incorporated into selenoproteins such as formate dehydrogenase (FDH), glycine reductase, and hydrogenase in bacteria as well as glutathione peroxidases, selenoprotein. SeryltRNASec , formed by the aminoacylation of tRNASec with serine by seryl-tRNA synthetase, is nucleophilically attacked by selenophosphate through the catalysis of selenocysteine synthase (SelA), resulting in selenocysteyl-tRNASec generation [9]. Sec is incorporated into selenoproteins at UGA codons [10,11,12,13]. The specific translation elongation factor, SelB, delivers selenocysteyl-tRNASec to the ribosome by recognizing the Sec insertion sequence (SECIS) located immediately downstream of the UGA codon on the mRNA. SeryltRNASec is further converted to O-phosphoseryl-tRNASec by O-phosphoseryl-tRNA Sec kinase, selenocysteyl-tRNASec is produced by Sep-tRNA:Sec-tRNA synthase using selenophosphate [14]. Mammalian SECIS is present in the 3’-untranslated region, and the SelB recognizes SECIS via a SECIS binding protein [6]

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Results

Conclusion