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.

Charcot‐Marie‐Tooth disease (CMT) is a genetically diverse hereditary disorder that affects the motor and sensory nerves, impacting about 1 in 2500 people. It can be inherited through autosomal dominant (AD), autosomal recessive (AR), or X‐linked genetic patterns. CMT2, one of the primary subtypes, is characterized by axonal degeneration and commonly presents with muscle weakness, atrophy, foot deformities, and sensory loss. Aminoacyl‐tRNA synthetases (aaRSs) play an important role in the genetic underpinnings of CMT2, with more than 60 disease‐causing alleles identified across eight different aaRSs, including alanyl‐, asparaginyl‐, histidyl‐, glycyl‐, methionyl‐, tryptophanyl‐, seryl‐, and tyrosyl‐tRNA synthetases. Mutations in aaRS genes can lead to destabilization of the enzyme, reduced aminoacylation, and aberrant protein complex formation. Yeast as a simple organism provides a robust model system to study the pathogenic effects of aaRS CMT mutations. In this review, we discuss the advantages and limitations of the yeast model systems for CMT2‐causative mutations in aaRS.

Dominant pathogenic mutations in tyrosyl-tRNA synthetase (YARS1) are associated with Charcot–Marie–Tooth disease (CMT), a progressive peripheral neuropathy for which no disease-modifying therapies currently exist. While recent advances in amino acid supplementation therapies suggest potential benefit for recessive aminoacyl-tRNA synthetase disorders, their applicability to dominant YARS1-associated neuropathies remains unclear. Here, we investigated the pathogenic mechanisms underlying the dominant YARS1 variants G41R, D81I, and E196Q. Using biochemical and functional analyses, we identified increased structural rigidity for G41R and E196Q proteins, while D81I is more susceptible to tryptic digestion. Furthermore, expression of the YARS1 variants in a humanized yeast model produced a dominant negative growth defect that is exacerbated at elevated temperatures, supporting disruption of canonical YARS1 function as a contributor to disease pathogenesis. Notably, tyrosine supplementation significantly rescued the observed growth defects across variants. These findings demonstrate that impaired tyrosine utilization contributes to the pathogenic effects of dominant YARS1 variants and provide proof-of-concept evidence that tyrosine supplementation may represent a potential therapeutic strategy for patients with YARS1-associated CMT.