Blocking S -adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing

Abstract

Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally modified eukaryotic proteins for x-ray crystallography. However, extensive replacement of methionine by selenomethionine for anomalous dispersion phasing has proven intractable in yeast. We report a general method to incorporate selenomethionine into proteins expressed in yeast based on manipulation of the appropriate metabolic pathways. sam1 ؊ sam2 ؊ mutants, in which the conversion of methionine to S-adenosylmethionine is blocked, exhibit reduced selenomethionine toxicity compared with wild-type yeast, increased production of protein during growth in selenomethionine, and efficient replacement of methionine by selenomethionine, based on quantitative mass spectrometry and x-ray crystallography. The structure of yeast tryptophanyl-tRNA synthetase was solved to 1.8 Å by using multiwavelength anomalous dispersion phasing with protein that was expressed and purified from the sam1 ؊ sam2 ؊ strain grown in selenomethionine. Six of eight selenium residues were located in the structure.x-ray crystallography ͉ methionine ͉ Saccharomyces cerevisiae

Fungal S-adenosylmethionine synthetase and the control of development and secondary metabolism in Aspergillus nidulans

“…As found for A. nidulans, yeast Sam1 also recruits proteins required for morphogenesis like the aconitase Aco1 or a-tubulin Tub1 (Gavin et al, 2006). In addition, the recruitment of the cobalamin-independent methionine synthetase Met6 (Malkowski et al, 2007) and the heat shock protein Ssa1 (Gong et al, 2009) seem to be conserved. Heat shock proteins are methylated in response to stress (Chiang et al, 1996).…”

Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance

“…4C, equivalent partial resistance is seen for the sam1⌬ or sam2⌬ single-deletion strains, and additive resistance was seen for the sam1⌬ sam2⌬ strain equivalent to the cys3⌬ strain up to 0.15 mM. Malkowski et al (9) demonstrated that deletion of the SAM1 gene resulted in greater resistance than deletion of the SAM2 gene. To determine the possible reasons for this difference, sam1⌬ and sam2⌬ single-mutant strains as well as a sam1⌬ sam2⌬ double-mutant strain in the BY4742 (MAT␣ MET15) were analyzed for SeMet resistance.…”

“…Thus, the single cys3⌬ deletion shows equivalent resistance to SeMet as the sam1⌬ sam2⌬ strain. Analysis of SeMet occupancy in a protein purified from the sam1⌬ sam2⌬ strain showed 83% occupancy in the presence of 0.125 mM SeMet and 95% occupancy in 0.5 mM SeMet (9). To compare the results for an expressed protein in the cys3⌬ and sam1⌬ sam2⌬ strain, a plasmid expressing 6ϫHis tagged S. cerevisiae eEF1B␣ was transformed into each strain.…”

“…To compare wildtype and cys3⌬ strains, some Met was required in the media to support growth of the wild-type strain. Thus, cells were grown in C-overnight, diluted to an OD 600 of 0.15, centrifuged, and the pellet resuspended in C-plus Cys plus 0.02 mM SeMet (9) demonstrated that deletion of the SAM1 and SAM2 genes encoding the AdoMet synthase resulted in additive enhanced resistance to SeMet. Resistance was seen following growth of the sam1⌬ sam2⌬ strain on solid C-media lacking Met or a sam1⌬ sam2⌬ met6⌬ strain on C-complete media with 0.06 mM SeMet.…”

“…However, although this approach works for a very highly expressed endogenous protein, because of the difficulty in applying molecular genetic approaches in industrial strains it is not applicable to conditions where the chromosomal gene encoding the protein must be deleted or the protein of interest must be expressed from a plasmid. Recent studies have determined that deletion of the 2 genes encoding encoding (S)-adenosylmethionine (AdoMet) synthase in S. cerevisiae results in enhanced tolerance of SeMet (9). This approach, however, requires the deletion of 2 genes, supplementation of the media with (S)-adenosyl methionine, and high concentrations of SeMet to achieve 95% occupancy.…”