Enniatin synthetase is a monomer with extended structure: evidence for an intramolecular reaction mechanism.
Enniatin synthetase (Esyn), a 347-kDa multienzyme consisting of two substrate activation modules, is responsible for the nonribosomal formation of the cyclohexadepsipeptide enniatin. The synthesis follows the so-called thiol template mechanism. While this process is basically well established, no substantial insight into the 3-dimensional arrangement of these enzymes and possible interactions between them exists to date. To find out whether enniatin synthesis is an intramolecular process or the result of three interacting Esyn molecules (intermolecular), analytical ultracentrifugation equilibration studies were carried out. The molecular mass of Esyn was determined by ultracentrifugation and is in good agreement with that calculated from the ORF of the encoding gene, indicating that Esyn exists in solution as a monomer. This strongly suggests that synthesis of the cyclohexadepsipeptide enniatin follows an intramolecular reaction mechanism in which all three reaction cycles are catalyzed by a single Esyn molecule. This finding was supported by in vitro complementation studies in which [(14)C]-methylvalyl Esyn, upon incubation with the second substrate D-2-hydroxyisovaleric acid (D-Hiv) and ATP, did not yield radioactive enniatin. This confirms our previous assumption of an iterative reaction mechanism similar to that for fatty acid synthase. Furthermore, the sedimentation rate constant evaluated from analytical ultracentrifugation was lower (S(20,w)=14.1S) than expected (S(20,w)=16.9S) for a globular protein, indicating that Esyn has an extended structure.
Glinski
Mirko
M
Fakultät II, Institut für Chemie, Arbeitsgruppe Biochemie und Molekulare Biologie, Technische Universität Berlin, Franklinstrasse 29, 10587 Berlin, Germany.
Urbanke
Claus
C
Hornbogen
Till
T
Zocher
Rainer
R
D000900:Anti-Bacterial Agents / Q000378:metabolism; D047630:Depsipeptides; D005670:Fusarium / Q000201:enzymology* / Q000254:growth & development; D010453:Peptide Synthases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides; D010456:Peptides, Cyclic / Q000378:metabolism; D014461:Ultracentrifugation
D000900:Anti-Bacterial Agents; D047630:Depsipeptides; D010455:Peptides; D010456:Peptides, Cyclic; C100264:enniatins; D010453:Peptide Synthases; C023122:enniatin B synthetase