Functional characterization of the recombinant N-methyltransferase domain from the multienzyme enniatin synthetase.
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.
Hornbogen
Till
T
Technische Universität Berlin, Fakultät II-Institut für Chemie, AG Biochemie und Molekulare Biologie, Franklinstrasse 29, 10997 Berlin, Germany.
Riechers
Sean-Patrick
SP
Prinz
Bianka
B
Schultchen
Jeffrey
J
Lang
Christine
C
Schmidt
Sebastian
S
Mügge
Clemens
C
Turkanovic
Suada
S
Süssmuth
Roderich D
RD
Tauberger
Eva
E
Zocher
Rainer
R
D002384:Catalysis; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology; D005285:Fermentation; D005670:Fusarium / Q000201:enzymology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008780:Methyltransferases / Q000737:chemistry*; D010453:Peptide Synthases / Q000737:chemistry*; D019920:Photoaffinity Labels; D011994:Recombinant Proteins / Q000737:chemistry; D012436:S-Adenosylmethionine; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000939:Epitopes; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D008780:Methyltransferases; D010453:Peptide Synthases; C023122:enniatin B synthetase