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Article (Scientific journals)
Functional characterization of the recombinant N-methyltransferase domain from the multienzyme enniatin synthetase.
Hornbogen, Till; Riechers, Sean-Patrick Hermann; Prinz, Bianka et al.
2007In Chembiochem : a European journal of chemical biology, 8 (9), p. 1048-54
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Keywords :
Catalysis; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Epitopes; Escherichia coli/enzymology; Fermentation; Fusarium/enzymology; Kinetics; Magnetic Resonance Spectroscopy; Methyltransferases/chemistry; Peptide Synthases/chemistry; Photoaffinity Labels; Recombinant Proteins/chemistry; S-Adenosylmethionine; Saccharomyces cerevisiae/enzymology; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Abstract :
[en] A 51 kDa fusion protein incorporating the N-methyltransferase domain of the multienzyme enniatin synthetase from Fusarium scirpi was expressed in Saccharomyces cerevisiae. The protein was purified and found to bind S-adenosyl methionine (AdoMet) as demonstrated by cross-linking experiments with (14)C-methyl-AdoMet under UV irradiation. Cofactor binding at equilibrium conditions was followed by saturation transfer difference (STD) NMR spectroscopy, and the native conformation of the methyltransferase was assigned. STD NMR spectroscopy yielded significant signals for H(2) and H(8) of the adenine moiety, H(1') of D-ribose, and S-CH(3) group of AdoMet. Methyl group transfer catalyzed by the enzyme was demonstrated by using aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) of L-Val, L-Ile, and L-Leu, which mimic the natural substrate amino acids of enniatin synthetase presented by the enzyme bound 4'-phosphopantetheine arm. In these experiments the enzyme was incubated in the presence of the corresponding aminoacyl-SNAC and (14)C-methyl-AdoMet for various lengths of time, for up to 30 min. N-[(14)C-Methyl]-aminoacyl-SNAC products were extracted with EtOAc and separated by TLC. Acid hydrolysis of the isolated labeled compounds yielded the corresponding N-[(14)C-methyl] amino acids. Further proof for the formation of N-(14)C-methyl-aminoacyl-SNACs came from MALDI-TOF mass spectrometry which yielded 23 212 Da for N-methyl-valyl-SNAC, accompanied by the expected postsource decay (PSD) pattern. Interestingly, L-Phe, which is not a substrate amino acid of enniatin synthetase, also proved to be a methyl group acceptor. D-Val was not accepted as a substrate; this indicates selectivity for the L isomer.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Hornbogen, Till
Riechers, Sean-Patrick Hermann  ;  Technische Universität Berlin, Fakultät II-Institut für Chemie, AG Biochemie und Molekulare Biologie, Franklinstrasse 29, 10997 Berlin, Germany.
Prinz, Bianka
Schultchen, Jeffrey
Lang, Christine
Schmidt, Sebastian
Mügge, Clemens
Turkanovic, Suada
Süssmuth, Roderich D.
Tauberger, Eva
Zocher, Rainer
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Title :
Functional characterization of the recombinant N-methyltransferase domain from the multienzyme enniatin synthetase.
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Chembiochem : a European journal of chemical biology
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Peer reviewed :
Peer reviewed
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