Purification and characterization of bimodular and trimodular derivatives of the erythromycin polyketide synthase.
Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. DEBS is a dimeric protein complex that consists of three large multidomain polypeptide chains, DEBS 1, DEBS 2, and DEBS 3. In turn, each polypeptide includes two modules, where one module is responsible for a single round of condensation and associated reduction reactions. A hybrid protein comprised of the first two modules of DEBS fused to a thioesterase domain (DEBS 1 + TE) was purified to homogeneity in a fully active form (Kcat = 4.8 min-1). Synthesis of the anticipated triketide lactone required the presence of (2RS)-methylmalonyl-CoA and NADPH. When available, propionyl-CoA is the preferred source of primer units. However, in its absence the enzyme can derive primer units via decarboxylation of a methylmalonyl extender. The two subunits of an engineered trimodular derivative of DEBS, DEBS 1 and module 3 of DEBS 2 linked to the TE domain (module 3 + TE), were also individually purified and reconstituted to produce the expected tetraketide lactone in vitro (Kcat = 0.23 min-1). The considerably lower specific activity of this trimodular PKS relative to its bimodular counterpart presumably reflects inefficient association between DEBS 1 and module 3 + TE. As expected, module 3 + TE could be efficiently cross-linked as a homodimer. In contrast, no cross-links were detectable between modules 2 and 3, even though biosynthesis of the tetraketide requires transient interactions to occur between these two modules. Since module 3 only contains the minimal set of active sites required in a module (a ketosynthase, an acyltransferase, and an acyl carrier protein domain) and is the first active unimodular protein to be purified to homogeneity, it represents an attractive target for future biophysical and structural studies.
Pieper
R
R
Department of Chemical Engineering, Stanford University, California 94305-5025, USA.
Gokhale
R S
RS
Luo
G
G
Cane
D E
DE
Khosla
C
C
D002474:Cell-Free System; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D004917:Erythromycin / Q000737:chemistry*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D013869:Thiolester Hydrolases / Q000302:isolation & purification / Q000378:metabolism
D003432:Cross-Linking Reagents; D009097:Multienzyme Complexes; D004917:Erythromycin; D013869:Thiolester Hydrolases