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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.

SUBMITTER: Malkowski MG

PROVIDER: S-EPMC1850019 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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

Malkowski Michael G MG Quartley Erin E Friedman Alan E AE Babulski Julie J Kon Yoshiko Y Wolfley Jennifer J Said Meriem M Luft Joseph R JR Phizicky Eric M EM DeTitta George T GT Grayhack Elizabeth J EJ

Proceedings of the National Academy of Sciences of the United States of America 20070410 16

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 ...[more]

PMID: 17426150

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Project description:Selenium (Se) is an essential trace element for humans and animals. Development and application of new forms of Se sources with lower toxicity and higher bioavailability has been attracting more attention. However, the bioavailabilities of Se from several new Se sources for broilers remain unclear. Therefore, the aim of this study was to assess the relative bioavailabilities of Se from Se yeast (SY), selenomethionine (SM), hydroxyl-selenomethionine (SO) and nano-Se (NS) relative to sodium selenite (SS) for broilers fed a conventional corn-soybean meal diet. A total of 576 one-day-old Arbor Acres commercial male broilers were randomly assigned to 16 treatments with 6 replicate cages per treatment in a completely randomized design involving a 5 (Se sources: SY, SM, SO, NS and SS) × 3 (added Se levels: 0.15, 0.30 and 0.45 mg Se/kg) factorial design of treatments plus 1 (a Se-unsupplemented control) for 21 d. The relative bioavailabilities of Se sources were estimated based on plasma or tissue Se concentrations as well as selenoprotein mRNA expressions and activities in broilers. The results showed that the Se concentrations and glutathione peroxidase (GPX) activities in plasma, liver, breast muscle, pancreas and kidney as well as Se concentration in erythrocytes of broilers, and Gpx1 and Selenop mRNA expressions in pancreas increased linearly (p < 0.03) as added Se level increased. Furthermore, the differences (p < 0.05) among different Se sources were detected for the Se concentrations in liver, breast muscle, pancreas and erythrocytes, GPX activities in pancreas and kidney. Based on slope ratios from the multiple linear regressions of the above indices, the Se bioavailabilities of SY, SM, SO, NS relative to SS (100%) were 78 to 367%, 67.8 to 471%, 57 to 372%, and 45 to 92%, respectively. The results from this study indicated that the Se from SM, SY and SO are more available to broilers than the Se from SS in enhancing the Se concentrations in liver, breast muscle, pancreas and erythrocytes and GPX activity in pancreas, and the Se from SM had the highest while the Se from NS had the lowest relative bioavailability.

Dataset Information

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

Publications

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

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