Enniatin synthetase, a novel type of multifunctional enzyme catalyzing depsipeptide synthesis in Fusarium oxysporum

Abstract

Enniatin synthetase, a multifunctional enzyme catalyzing depsipeptide formation in Fusarium oxysporum was purified to 98% homogeneity as judged by analytical disc gel electrophoresis. The enzyme consists of a single polypeptide chain of a molecular weight of about 250 000. Similar to a number of peptide synthetases and to fatty acid synthetase the enzyme contains 4'-phosphopantetheine as a prosthetic group. Studies on substrate specificity revealed that the enzyme is capable of synthesizing enniatins A--C and also mixed-type enniatins containing more than one species of amino acid. A linear dependence of rate of enniatin synthesis on enzyme concentration was observed, indicating that depsipeptide formation is an intramolecular process. Omission of the methyl donor S-adenosyl-L-methionine resulted in the formation of unmethylated enniatins with a reaction rate of about 10% of that observed in the case of enniatins. Sulfhydryl-directed reagents generally had an inhibitory influence on enniatin synthesis. However, inhibition studies with iodoacetamide revealed that it behaves differently toward the hydroxy acid site(s) and amino acid site(s). This indicates the presence of chemically distinct thiol groups in the active sites for the two substrates.

Fungal cyclooligomerdepsipeptides: From classical biochemistry to combinatorial biosynthesis

“…84 This has been confirmed for all other CODSs studied to date, 61,79 and can also be concluded independently from their identical domain organization. Substrate requirements reveal the overall reaction 3 AA + 3 HA + 3 SAM + 6 ATP / COD + 6 AMP + 6 PP i + 3 SAH (1) where…”

“…The specific protocols varied with the strains used, the properties of the mycelia, the modes of cell disruption, and the enzyme content. Detailed protocols were established for enniatin synthetases from Fusarium equiseti (synonym: F. scirpi, previously described as F. oxysporum), 79 F. sambucinum and F. lateritium; 81 for the beauvericin synthetase from Beauveria bassiana; 82 and for the PF1022 synthetase from Mycelia sterilia (Rosellinia sp.). 61 These CODS have been purified to homogeneity as judged by SDS polyacrylamide electrophoresis, and their molecular masses have been estimated by gel filtration, sucrose gradient ultracentrifugation and electrophoretic mobilities.…”

“…79 In an extended approach, Zocher and coworkers used additional amino acid substrate analogs for the incorporation of amino acids L-Ala, L-Cys, L-Thr and L-Ser into the enniatin structure (Fig. 8A).…”

D-Lysergyl Peptide Synthetase from the Ergot Fungus Claviceps purpurea

“…Detection of immunopositive bands was performed by standard techniques using 1:10,000 dilution of primary antibody, phosphatase-conjugated goat anti-rabbit antibody (Sigma) and nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as reagents. 4Ј-Phosphopantetheine determinations of protein fractions (liquid samples) were done by the method of Pugh and Wakil (22) modified according to Zocher et al (23). Alternatively determinations of 4Ј-phosphopantetheine in protein bands were done according to a method of Stindl (24).…”

Biosynthesis of PF1022A and Related Cyclooctadepsipeptides

“…Enniatins are synthesized by the multifunctional enzyme ESYN, which was the first N-methyl-cyclopeptide synthetase ever characterized. Biochemical investigations revealed that this enzyme is one single polypeptide chain with a molecular mass of about 350 kDa (8,9). All catalytic functions necessary for enniatin synthesis, i.e.…”