Use of protein trans-splicing to produce active and segmentally 2H, 15N labeled mannuronan C5-epimerase AlgE4

Edith Buchinger, Finn L. Aachmann, Sesilja Aranko, Svein Valla, Gudmund Skjåk-BræK, Hideo Iwaï, Reinhard Wimmer*

*Tämän työn vastaava kirjoittaja

Tutkimustuotos: LehtiartikkeliArticleScientificvertaisarvioitu

11 Sitaatiot (Scopus)

Abstrakti

Alginate epimerases are large multidomain proteins capable of epimerising C5 on β-D-mannuronic acid (M) turning it into α-L-guluronic acid (G) in a polymeric alginate. Azotobacter vinelandii secretes a family of seven epimerases, each of which is capable of producing alginates with characteristic G distribution patterns. All seven epimerases consist of two types of modules, denoted A and R, in varying numbers. Attempts to study these enzymes with solution-state NMR are hampered by their size - the smallest epimerase, AlgE4, consisting of one A- and one R-module, is 58 kDa, resulting in heavy signal overlap impairing the interpretation of NMR spectra. Thus we obtained segmentally 2H, 15N labeled AlgE4 isotopomeres (A-[ 2H, 15N]-R and [ 2H, 15N]-A-R) by protein trans-splicing using the naturally split intein of Nostoc punctiforme. The NMR spectra of native AlgE4 and the ligated versions coincide well proving the conservation of protein structure. The activity of the ligated AlgE4 was verified by two different enzyme activity assays, demonstrating that ligated AlgE4 displays the same catalytic activity as wild-type AlgE4. Published by Wiley-Blackwell.

AlkuperäiskieliEnglanti
Sivut1534-1543
Sivumäärä10
JulkaisuProtein Science
Vuosikerta19
Numero8
DOI - pysyväislinkit
TilaJulkaistu - elok. 2010
OKM-julkaisutyyppiA1 Julkaistu artikkeli, soviteltu

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