Biosynthesis by Escherichia coli of active altered proteins containing selenium instead of sulfur

Cowie, Dean B.; Cohen, Georges N.

doi:10.1016/0006-3002(57)90003-3

Cited by 273 publications

(157 citation statements)

References 12 publications

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“…However, the catalytic capacity, as measured by V,,,, unaltered (Table 3). Therefore, these findings do not contradict the initial observation of Cowie and Cohen (1957) that selenomethionine-containing proteins are fully functional. In further analyses, conducted in Hepes rather than Tris due to the effect of Tris buffers on the K,, we also noticed a different response in metal inhibition studies.…”

Section: Resultssupporting

confidence: 58%

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Selenomethionine Labelling of Phosphomannose Isomerase Changes its Kinetic Properties

Bernard¹,

Wells²,

Cleasby³

et al. 1995

European Journal of Biochemistry

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Phosphomannose isomerase (PMI) is an essential enzyme in the early steps of the protein glycosylation pathway in both prokaryotes and eukaryotes. Lack of the enzyme is lethal for fungal organisms and it is thus a potential fungicidal target. To facilitate the solution of the three-dimensional structure of the enzyme from the pathogen Candida albicans, we have produced the recombinant selenomethionine-labelled enzyme (SeMet-PMI). D U l , a methionine auxotroph Escherichia coli strain, was transformed with a PMI expression plasmid and grown on an enriched selenomethionine-containing medium to highcell densities. The SeMet-PMI protein has been purified and found by amino acid analysis to have its methionine residues replaced by selenomethionine residues. Electrospray mass spectroscopy showed a major species of 49063 ? 10 Da for SeMet-PMI compared to 48735 ? 6 Da for the normal recombinant enzyme, accounting for the incorporation of seven selenomethionine residues. SeMet-PMI crystallised isomorphously with the normal PMI protein and the crystals diffract to 0.23 nm. Kinetic characterisation of SeMet-PMI showed that its K, for the substrate mannose-6-phosphate was fourfold higher than that of its methionine-containing counterpart. The inhibition constant for zinc ions was also increased by a similar factor. However, the V,, was unaltered. These results suggested that one or more methionine residues must be in close proximity to the substrate-binding pocket in the active sire, rendering substrate access more difficult compared to the normal enzyme. This hypothesis was confirmed by the finding of four methionine residues lying along one wall of the active site.

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Section: Resultssupporting

confidence: 58%

“…controlled incorporation of selenium into proteins by replacement of the other sulphur-containing amino acid, methionine, became feasible through the work of Cowie and Cohen (1957). They isolated a methionine auxotroph from Escherichia coli which was fully viable when grown in the presence of selenomethionine as sole source of methionine.…”

mentioning

confidence: 99%

Selenomethionine Labelling of Phosphomannose Isomerase Changes its Kinetic Properties

Bernard¹,

Wells²,

Cleasby³

et al. 1995

European Journal of Biochemistry

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“…The ability of E. coli to incorporate nonproteinogenic amino acids into polypeptides has been known for decades (Cowie and Cohen, 1957;Fenster and Anker, 1969;Hagen et al, 1978;Hagen et al, 1979;Rennert and Anker, 1963), but this property has only recently been exploited for the modification of recombinant proteins. The two main strategies are the residue-specific incorporation of nonnatural amino acids by misaminoacylation of transferRNAs (tRNAs) (Kiick et al, 2001;Kirshenbaum et al, 2002;Niemz and Tirrell, 2001) and the site-directed incorporation, which utilizes non-cognate amber suppressor tRNA/aminoacyl-tRNA synthetase pairs (Noren et al, 1989;Wang et al, 2001).…”

Section: Biosynthetic Methods For Incorporation Of Novel Amino Acidsmentioning

confidence: 99%

Gelatinase-mediated migration and invasion of cancer cells

Björklund

Koivunen

2005

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer

463

609

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“…I published with Halvorson and Spiegelman a correction to their previous statement (13). Dean Cowie and I showed that, in contrast to the analogs causing linear growth and not allowing the growth of the corresponding auxotrophic mutants, the addition of selenomethionine, in which the sulfur atom of methionine is replaced by a selenium atom, allowed the exponential growth of a methionine-requiring mutant in the absence of methionine (12,25). More than thirty years after our publication, I had the privilege to present a paper by Wayne Hendrickson to the EMBO Journal (30): He had taken advantage of our observation to study the threedimensional structure of proteins in which methionine was replaced by selenomethionine, and solve the phase problem at the same time.…”

Section: Wwwannualreviewsorg • Looking Backmentioning

confidence: 99%

Looking Back

Cohen

2005

Annu. Rev. Microbiol.

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My encounter with Jacques Monod has shaped my scientific career. After a short incursion in the biochemistry of strict anaerobes, and after elucidating the biosynthetic pathway leading from aspartate to threonine in Escherichia coli, I joined his laboratory. With him and Howard Rickenberg, I discovered the stereospecific permeability of galactosides and amino acids (permeases). After this intermezzo, I returned to the analysis of biosynthetic pathways and of their regulation by allosteric feedback inhibition and repression in E. coli. Among others, my studies led to the discovery of the tryptophan and methionine repressors, to the incorporation of amino acid analogues in proteins, including selenomethionine (which much later led to progress in protein crystallography), to the definition of isofunctional and multifunctional enzymes, and to the elucidation of the primary structure of most of the enzymes leading to threonine and methionine.

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Biosynthesis by Escherichia coli of active altered proteins containing selenium instead of sulfur

Cited by 273 publications

References 12 publications

Selenomethionine Labelling of Phosphomannose Isomerase Changes its Kinetic Properties

Selenomethionine Labelling of Phosphomannose Isomerase Changes its Kinetic Properties

Gelatinase-mediated migration and invasion of cancer cells

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