Architectures of whole-module and bimodular proteins from the 6-deoxyerythronolide B synthase.
The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase produced by the actinomycete Saccharopolyspora erythraea that synthesizes the macrocyclic core of the antibiotic erythromycin 6-deoxyerythronolide B. The megasynthase is a 2-MDa trimeric complex composed of three unique homodimers assembled from the gene products DEBS1, DEBS2, and DEBS3, which are housed within the erythromycin biosynthetic gene cluster. Each homodimer contains two clusters of catalytically independent enzymatic domains, each referred to as a module, which catalyzes one round of polyketide chain extension and modification. Modules are named sequentially to indicate the order in which they are utilized during synthesis of 6-deoxyerythronolide B. We report small-angle X-ray scattering (SAXS) analyses of a whole module and a bimodule from DEBS, as well as a set of domains for which high-resolution structures are available. In all cases, the solution state was probed under previously established conditions ensuring that each protein is catalytically active. SAXS data are consistent with atomic-resolution structures of DEBS fragments. Therefore, we used the available high-resolution structures of DEBS domains to model the architectures of the larger protein assemblies using rigid-body refinement. Our data support a model in which the third module of DEBS forms a disc-shaped structure capable of caging the acyl carrier protein domain proximal to each active site. The molecular envelope of DEBS3 is a thin elongated ellipsoid, and the results of rigid-body modeling suggest that modules 5 and 6 stack collinearly along the 2-fold axis of symmetry.
Department of Chemistry, Stanford University, Stanford, CA 94305, USA. Electronic address: edwardal@stanford.edu.
Andrea L
AL
Edwards
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, 14 2575 Sand Hill Road, MS69, Menlo Park, CA 94025, USA.
Tsutomu
T
Matsui
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, 14 2575 Sand Hill Road, MS69, Menlo Park, CA 94025, USA.
Thomas M
TM
Weiss
Department of Chemistry, Stanford University, Stanford, CA 94305, USA; Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA; Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
Chaitan
C
Khosla
D000595:Amino Acid Sequence; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048630:Polyketide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D016930:Saccharopolyspora / Q000201:enzymology* / Q000235:genetics; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
D009097:Multienzyme Complexes; D048630:Polyketide Synthases
6-deoxyerythronolide B synthase; small-angle X-ray scattering; type I polyketide synthase