PMC

A. Chemical structure of selenoneine. B. Results of metabolomic analysis of WT and P3nmt1-egt1⁺ strains in EMM2+Se and EMM2-N+Se media. Normalized peak areas of EGT and selenoneine are shown. C. Extracted ion chromatograms of EGT and selenoneine masses in raw LC-MS data acquired from metabolome samples of nitrogen-starved cells (24 h in EMM2-N+Se medium) of WT and Δegt1 strains. Note that the intensity scale of the selenoneine plot is 1% relative to that of the EGT plot. D. Extracted ion chromatograms of EGT and selenoneine masses in raw LC-MS data acquired from metabolome samples of WT and Δegt1 strains cultivated in EMM2+Se medium with and without supplementation with 1 mM pure EGT. Note that the intensity scale of the selenoneine plot is 0.1% relative to that of the EGT plot. E. Extracted ion chromatograms of six compound masses in WT, P3nmt1-egt1⁺, and P3nmt1-egt1⁺ Δegt2 strains. The plots of hercynylcysteine and hercynylselenocysteine sulfoxide show mass values calculated from their predicted chemical formulas (C₁₂H₂₁N₄O₄S⁺  = 317.128 m/z for hercynylcysteine, and C₁₂H₂₁N₄O₅Se⁺  = 381.067 m/z for hercynylselenocysteine sulfoxide, respectively). Note that the intensity scale of the plots in the right half of the figure is adjusted to 10% relative to the plots in the left half of the figure.

A and B. A scatter plot comparing results of metabolomic analysis of WT vs. Δegt1 (A) or Δegt2 (B) strains under nitrogen starvation (24 h in EMM2-N medium). Each dot represents a single identified metabolite. Values on both scales indicate normalized peak areas of metabolite peaks in corresponding strains. Red diagonal lines indicate a 2-fold difference. C. Results of metabolomic analysis of WT and egt1⁺ overexpression strains. Cells were cultivated at 26°C in the EMM2 medium lacking thiamine for at least 24 h. Cultures indicated +Thiamine were cultivated in the presence of 5 µg/ml thiamine for 24 h. Normalized peak areas of compounds composing the EGT pathway are shown. D. Time course metabolomic analysis of quiescent S. pombe cultures under nitrogen (EMM2-N) and glucose (EMM2-LG) starvation. Values represent means ± standard deviations of normalized peak areas of EGT in three independent cell cultures. E. Time course viability results of WT and Δegt1 deletion mutant cultures under nitrogen (EMM2-N) and glucose (EMM2-LG) starvation.

A. Previously published EGT biosynthesis pathways in M. smegmatis and N. crassa. The Egt-1 protein in N. crassa is a fusion enzyme that catalyzes two different reactions. B. Comparison of the conserved domain structure of S. pombe protein SPBC1604.01 (designated Egt1 in this manuscript) with S. japonicus SJAG_00832 and EGT biosynthesis proteins in N. crassa (Egt-1) and M. smegmatis (EgtD and EgtB). All proteins are composed of: 1. an S-adenosyl-L-methionone (SAM)-dependent methyltransferase domain, including DUF2260, a domain of unknown function; 2. an uncharacterized DinB_2 domain, including an iron-binding motif HX₃HXE; and 3. a formylglycine generating enzyme (FGE)-sulfatase domain. Percentage identity (% id) of amino acid sequences is indicated in comparison to the corresponding sequence in S. pombe. C. Comparison of conserved domain structure of M. smegmatis protein EgtE with its four putative homologs in S. pombe. All proteins contain a single pyridoxal phosphate (PLP)-binding cysteine desulfurase domain. The conserved catalytic residues (PLP binding sites) are indicated by red lines. Percentage identity (% id) of the amino acid sequences is indicated in comparison to the corresponding sequence in M. smegmatis. D. Phylogenetic tree visualizing the similarity of amino acid sequences of M. smegmatis EgtE and its putative homologs in S. pombe. E. Normalized peak areas of hercynylcysteine sulfoxide and EGT obtained by metabolomic analysis of WT and deletion mutant S. pombe strains. Cells were nitrogen-starved prior to analysis (24 h in EMM2-N medium) to induce EGT synthesis.