Monoclonal antibodies to the multienzyme enniatin synthetase. Production and use in structural studies.
Monoclonal antibodies have been prepared against the multifunctional enzyme enniatin synthetase, which catalyses the biosynthesis of the cyclodepsipeptide antibiotic enniatin. Five different antibodies (designated 1.56, 21.1, 25.91, 28.7 and 28.34) were characterized. 1.56, 21.1 and 25.91 were of IgG1 and 28.7 and 28.34 of IgM subclass. Binding studies showed that 21.1 and 25.91 are obviously directed against determinants based on the primary structure of the enzyme, whereas 28.7, 28.34 and 1.56 bind to the native enzyme. All antibodies inhibited enniatin formation. Based on their ability to inhibit different partial reactions of the multienzyme the antibodies could be divided into three groups: 21.1 and 25.91 inhibit valyl thioester formation, 1.56 additionally inhibits D-2-hydroxyisovaleric acid thioesterification, and 28.7 and 28.34 block both thioester sites as well as the N-methylation step. None of the antibodies affected the formation of L-valyl or D-hydroxyisovaleryl adenylate by the enzyme. The results indicate that there must be distinct thioester activation sites for valine and D-hydroxyisovalerate close to each other and in the neighbourhood of the methyltransferase site. The adenylation sites for D-hydroxy-isovalerate and L-valine are obviously located at some distance.
D000345:Affinity Labels; D000818:Animals; D000900:Anti-Bacterial Agents; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000302:isolation & purification; D000918:Antibody Specificity; D003429:Cross Reactions; D047630:Depsipeptides; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007457:Iodine Radioisotopes; D051379:Mice; D008807:Mice, Inbred BALB C; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000302:isolation & purification; D010455:Peptides; D010456:Peptides, Cyclic / Q000096:biosynthesis
D000345:Affinity Labels; D000900:Anti-Bacterial Agents; D000911:Antibodies, Monoclonal; D047630:Depsipeptides; D007457:Iodine Radioisotopes; D010455:Peptides; D010456:Peptides, Cyclic; C100264:enniatins; D010453:Peptide Synthases; C023122:enniatin B synthetase