title: 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 A 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.
authors.affiliation: Center for Pediatric Biomedical Research and Departments of Environmental Medicine and Biochemistry and Biophysics, University of Rochester Medical School, Rochester, NY 14642, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Malkowski
authors.forename: Erin
authors.initials: E
authors.lastname: Quartley
authors.forename: Alan E
authors.initials: AE
authors.lastname: Friedman
authors.forename: Julie
authors.initials: J
authors.lastname: Babulski
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Kon
authors.forename: Jennifer
authors.initials: J
authors.lastname: Wolfley
authors.forename: Meriem
authors.initials: M
authors.lastname: Said
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Luft
authors.forename: Eric M
authors.initials: EM
authors.lastname: Phizicky
authors.forename: George T
authors.initials: GT
authors.lastname: DeTitta
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Grayhack
mesh_terms: D018360:Crystallography, X-Ray; D012436:S-Adenosylmethionine / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D012645:Selenomethionine / Q000737:chemistry / Q000493:pharmacokinetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D012436:S-Adenosylmethionine; D012645:Selenomethionine