Efficient purification and kinetic characterization of a bimodular derivative of the erythromycin polyketide synthase.
Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are giant multienzymes that biosynthesize a number of clinically important natural products. The modular nature of PKSs suggests the possibility of a combinatorial approach to the synthesis of novel bioactive polyketides, but the efficacy of such a strategy depends critically on gaining fundamental insight into PKS structure and function, most directly through experiments with purified PKS proteins. Several recent investigations into important aspects of the activity of these enzymes have used only partially purified proteins (often 3-4% of total protein), reflecting how difficult it is to purify these multienzymes in amounts adequate for kinetic and structural analysis. We report here the steady-state kinetic analysis of a typical bimodular PKS, 6-deoxyerythronolide B synthase 1-thioesterase (DEBS 1-TE), purified from recombinant Saccharopolyspora erythraea JCB101 by a new, high-yielding procedure consisting of three steps: ammonium sulfate precipitation, hydrophobic interaction chromatography and size-exclusion chromatography. The method provides 13-fold purification with a recovery of 11% of the applied PKS activity. The essentially homogeneous synthase exhibits an intrinsic methylmalonyl-CoA hydrolase activity, which competes with polyketide chain extension. The most reliable value for the kcat for synthesis of (3S,5R)-dihydroxy-(2R,4R)-dimethyl-n-heptanoic acid-delta-lactone is 0.84 min-1, and the apparent Km for (2RS)-methylmalonyl-CoA is 17 microM. This kcat is approximately 10-fold lower than the value reported previously for a differently engineered version of the truncated PKS, DEBS 1+TE. The difference likely reflects the fact that the DEBS 1-TE contains a hybrid acyl carrier protein (ACP) domain in its second module, which lowers its catalytic efficiency.
Bycroft
M
M
Cambridge Centre for Molecular Recognition, Department of Organic Chemistry, University of Cambridge, UK.
Weissman
K J
KJ
Staunton
J
J
Leadlay
P F
PF
D000214:Acyl Coenzyme A / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism*; D011753:Pyrones / Q000378:metabolism; D016930:Saccharopolyspora / Q000737:chemistry / Q000201:enzymology*
C096704:2,4-dimethyl-3,5-dihydroxy-n-heptanoic acid delta lactone; D000214:Acyl Coenzyme A; D009097:Multienzyme Complexes; D011753:Pyrones; C015357:methylmalonyl-coenzyme A