Bacterial expression of catalytically active fragments of the multifunctional enzyme enniatin synthetase.
Enniatin synthetase catalyzes the biosynthesis of N-methylated cyclohexadepsipeptides. The 347 kDa enzyme is encoded by the esyn1 gene of Fusarium scirpi and contains two domains (EA and EB) homologous to each other and to regions of other microbial peptide synthetases. Parts of the esyn1 gene were subcloned in frame to a small lacZ gene portion of Escherichia coli expression vectors. Overproduced recombinant proteins showed a high tendency towards inclusion body formation and could be only partially dissolved in 8 M urea or 6 M guanidine hydrochloride. After renaturation, a 121 kDa recombinant protein representing the N-terminal conserved domain EA of enniatin synthetase was shown to activate D-hydroxyisolvaleric acid via adenylation. Similarly, a 158 kDa recombinant protein comprising the C-terminal conserved domain EB catalyzed the activation of the substrate amino acid (e.g. L-valine). Moreover, this protein could be photolabeled with S-[methyl-14C]adenosyl-L-methionine, (AdoMet) indicating the presence of the methyltransferase. Both functions, L-valine activation and AdoMet binding, could be assigned to a 108 kDa recombinant protein encompassing the A and the M segment of domain EB. The fact that a 65 kDa recombinant protein representing the M portion could be photolabeled, indicated the localization of the methyltransferase in this region. Three deletion mutants of the 65 kDa protein were shown to be inactive with respect to UV-induced AdoMet labeling.
Haese
A
A
Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Federal Republic of Germany.
Pieper
R
R
von Ostrowski
T
T
Zocher
R
R
D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli; D005670:Fusarium / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000378:metabolism; D010453:Peptide Synthases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010777:Photochemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014631:Valerates / Q000378:metabolism; D014633:Valine / Q000378:metabolism
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014631:Valerates; C035864:2-hydroxyisovaleric acid; D010453:Peptide Synthases; C023122:enniatin B synthetase; D014633:Valine