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. 2019 Aug 23;294(34):12855-12865.
doi: 10.1074/jbc.RA119.008219. Epub 2019 Jul 11.

A cysteinyl-tRNA synthetase variant confers resistance against selenite toxicity and decreases selenocysteine misincorporation

Kyle S Hoffman  1 , Oscar Vargas-Rodriguez  1 , Daniel W Bak  2 , Takahito Mukai  1 , Laura K Woodward  1 , Eranthie Weerapana  2 , Dieter Söll  1   3 , Noah M Reynolds  4
Affiliations

A cysteinyl-tRNA synthetase variant confers resistance against selenite toxicity and decreases selenocysteine misincorporation

Kyle S Hoffman et al. J Biol Chem. .

Abstract

Selenocysteine (Sec) is the 21st genetically encoded amino acid in organisms across all domains of life. Although structurally similar to cysteine (Cys), the Sec selenol group has unique properties that are attractive for protein engineering and biotechnology applications. Production of designer proteins with Sec (selenoproteins) at desired positions is now possible with engineered translation systems in Escherichia coli However, obtaining pure selenoproteins at high yields is limited by the accumulation of free Sec in cells, causing undesired incorporation of Sec at Cys codons due to the inability of cysteinyl-tRNA synthetase (CysRS) to discriminate against Sec. Sec misincorporation is toxic to cells and causes protein aggregation in yeast. To overcome this limitation, here we investigated a CysRS from the selenium accumulator plant Astragalus bisulcatus that is reported to reject Sec in vitro Sequence analysis revealed a rare His → Asn variation adjacent to the CysRS catalytic pocket. Introducing this variation into E. coli and Saccharomyces cerevisiae CysRS increased resistance to the toxic effects of selenite and selenomethionine (SeMet), respectively. Although the CysRS variant could still use Sec as a substrate in vitro, we observed a reduction in the frequency of Sec misincorporation at Cys codons in vivo We surmise that the His → Asn variation can be introduced into any CysRS to provide a fitness advantage for strains burdened by Sec misincorporation and selenium toxicity. Our results also support the notion that the CysRS variant provides higher specificity for Cys as a mechanism for plants to grow in selenium-rich soils.

Keywords: Astragalus bisulcatus; Escherichia coli (E. coli); aminoacyl tRNA synthetase; cysteinyl-tRNA synthetase; protein engineering; selenite toxicity; selenium; selenocysteine; transfer RNA (tRNA); translation.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Selenium metabolic pathways in E. coli and S. cerevisiae that lead to free Sec. Pathways specific for S. cerevisiae and E. coli are shown in red and blue, respectively. 1, in S. cerevisiae, SeMet is imported through amino acid permeases (30), whereas selenite is taken up through phosphate transporters and a monocarboxylate transporter (57, 58). 2, SeMet enters the Met metabolic cycle and is converted to Se-adenosylmethionine (SeAM) by SAM synthase (33). 3, the Met salvage pathway converts Se-adenosylmethionine back to SeMet. Se-adenosylmethionine is also a substrate of methyltransferases, which release Se-adenosylhomocysteine (SeAH) as a product of methylation reactions. Se-adenosylhomocysteine is hydrolyzed to selenohomocysteine (SeHCys) by S-adenosyl-l-homocysteine hydrolase. Selenohomocysteines can be methylated back to SeMet by Met synthase (59). 4, enzymes of the trans-sulfuration pathway convert selenohomocysteine to Sec (30). Cystathionine synthases and cystathionine lyases interconvert selenohomocysteine and selenocystathionine (SeCyst). Selenium from selenite can also replace sulfur in the trans-sulfuration pathway. 5, reduction of selenite to selenide occurs nonenzymatically and involves GSH and other organic selenium intermediates (60). 6, selenide reacts with O-acetylhomoserine in yeast to form selenohomocysteine. In E. coli, selenide can react with SAM to produce methylselenol (MSe) (61) (7), or it can be a substrate of O-acetylserine sulfhydrylases AB (cysM and cysK) for the production of free Sec (60, 62) (8). 9, because methionyl-tRNA synthetase and CysRS cannot discriminate between sulfur- and selenium-containing amino acids, SeMet and Sec are ligated onto tRNA and misincorporated at Met and Cys codons, respectively, during protein synthesis (figure was adapted from Ref. 59).
Figure 2.
Figure 2.
Position and conservation of E. coli CysRS-H235. A, E. coli CysRS crystal structure (Protein Data Bank (PDB) code 1LI7) showing the active-site residues involved in zinc coordination and substrate specificity. The highly conserved His-235 residue is shown in yellow, other conserved residues involved in substrate specificity are in purple, the zinc ion is shown as a silver sphere, the Cys substrate is in cyan, and two threonine residues that interact with the amino and carboxyl groups of Cys are in green. Hydrogen bonds are shown as yellow dashed lines. To show His-235, residues Asp-229 and Met-231 are represented as a dotted backbone. B, multiple sequence alignment of a portion of the CysRS catalytic-site residues from species spanning the tree of life. Conserved residues important for substrate specificity and zinc coordination are indicated with an asterisk. The Asn-240 variation in A. bisulcatus CysRS sequence is indicated by the arrow.
Figure 3.
Figure 3.
Growth curves of CysRS strains with increasing concentrations of sodium selenite. A, E. coli UQ818 strains expressing either empty pGEX-2T (control), cysS, cysS-H235N, or cysS-H235A were grown at 30 °C to stationary phase in LB medium containing 100 μg/ml ampicillin. Each strain was then diluted into the same medium with the indicated concentrations of sodium selenite and grown at 42 °C for 12 h. B, all strains used for growth curves in A were also grown at 30 °C at the indicated sodium selenite concentrations for 12 h. All error bars represent the standard deviation from three replicates.
Figure 4.
Figure 4.
Growth of S. cerevisiae CysRS strains in the presence of SeMet. Yeast strains BY4741 (WT) and YNL24YW deletion strains expressing either CysRS or CysRS-H395N from pRS313 were grown to stationary phase in SC medium containing 65 μm Met. 10-Fold serial dilutions of each strain were spotted onto either a YPD plate or SC medium plates containing the indicated amounts of Met and SeMet. The YPD plate was grown for 2 days, the SC plate with 65 μm Met was grown for 3 days, and the SC plate with 32.5 μm Met was grown for 4 days at 30 °C.
Figure 5.
Figure 5.
Chemoproteomic detection of misincorporated Sec in E. coli CysRS strains. A, general proteomic strategy for the detection of Sec-containing peptides through low-pH isoTOP-ABPP labeling of reactive thiols/selenols with isotopic iodoacetamide-alkyne probes. B, spectral count data for Cys (orange) and Sec (purple) peptides identified in the cysS (cysS, +Se) or cysS-H235N (cysS-H235N, +Se) strain grown in the presence of 1 mm selenite or the cysS strain grown in the absence of selenite (cysS, −Se). C, L/H ratio plot for Sec-containing peptides where ratios are indicative of the difference in selenopeptide abundance between the cysS (light) and cysS-H235N (heavy) strains. The median L/H ratio is 4.3. D, same as C for Cys-containing peptides. The median L/H ratio is 1.4. E, comparison of L/H ratios for quantified peptides that were identified in both the Cys (orange)- and Sec (purple)-containing forms. Extracted ion chromatograms and isotopic envelopes for both the Cys- and Sec-containing versions of three representative peptides are shown: cmoA, tRNA (cmo5U34) methyltransferase (NIHHDNC*K); ldhA, d-lactate dehydrogenase (GETC*PNELV); and clpB, chaperone protein ClpB (GELHC*VGATTLDEYR). Error bars in B and E represent standard deviation from two replicates. The double asterisks in B represent a significant difference in selenopeptide spectral counts as determined by a paired student's t-test (two-tailed), **, p < 0.01; ns, not significant.
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