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K J Weissman

Publications and source records attributed to K J Weissman.

6 recordsLinked to original sources
PubMed2000

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.

Acyl Coenzyme A↗
PubMed1998

Origin of starter units for erythromycin biosynthesis.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are multifunctional proteins that govern the synthesis of a number of clinically important natural products. The modular arrangement of active sites within these enzymes suggests the possibility of a combinatorial approach to the synthesis of novel bioactive polyketides. The efficacy of combinatorial strategies toward altering the starter unit specificity of polyketide synthases critically depends on controlling the supply of competing endogenous starter acids. Using DEBS 1-TE, a bimodular derivative of DEBS, we aimed to determine whether the beta-ketosynthase (KS) domain responsible for condensation in the first module also has the ability to prime its own biosynthesis by catalyzing the decarboxylation of methylmalonyl-CoA to produce propionyl-CoA. In contrast to earlier reports with a closely similar mini-PKS DEBS 1+TE, we have found that rigorously purified DEBS 1-TE does not catalyze the decarboxylation of methylmalonyl-CoA.

Acyl Coenzyme A↗
PubMed1998

Evaluating precursor-directed biosynthesis towards novel erythromycins through in vitro studies on a bimodular polyketide synthase.

BACKGROUND: Modular polyketide synthases (PKSs) catalyse the biosynthesis of complex polyketides using a different set of enzymes for each successive cycle of chain extension. Directed biosynthesis starting from synthetic diketides is a potentially valuable route to novel polyketides. We have used a purified bimodular derivative of the erythromycin-producing polyketide synthase (DEBS 1-TE) to study chain extension starting from a variety of diketide analogues and, in some cases, from the alternative acyl-CoA thioester substrates. RESULTS: Chain initiation in vitro by DEBS 1-TE module 2 using a synthetic diketide analogue as a substrate was tolerant of significant structural variation in the starter unit of the synthetic diketide, but other changes completely abolished activity. Interestingly, a racemic beta-keto diketide was found to be reduced in situ on the PKS and utilised in place of its more complex hydroxy analogue as a substrate for chain extension. The presence of a diketide analogue strongly inhibited chain initiation via the loading module. Significantly higher concentrations of diketide N-acetylcysteamine analogues than their corresponding acyl-CoA thioesters are required to achieve comparable yields of triketide lactones. CONCLUSIONS: Although a broad range of variation in the starter residue is acceptable, the substrate specificity of module 2 of a typical modular PKS in vitro is relatively intolerant of changes at C-2 and C-3. This will restrict the usefulness of approaches to synthesise novel erythromycins using synthetic diketides in vivo. The use of synthetic beta-keto diketides in vivo deserves to be explored.

Catalysis↗
PubMed1997

The molecular basis of Celmer's rules: the stereochemistry of the condensation step in chain extension on the erythromycin polyketide synthase.

Modular polyketide synthases (PKSs), for example, the 6-deoxyerythronolide B synthase (DEBS) responsible for synthesis of the aglycone core of the macrolide antibiotic erythromycin, generate an impressive diversity of asymmetric centers in their polyketide products. However, as noted by Celmer, macrolides have the same absolute configuration at all comparable stereocenters. Understanding how the stereochemistry of chain extension in controlled is therefore crucial to determining the common mechanism of action of these enzymes. We aimed to elucidate the molecular basis of Celmer's rules through in vitro studies with DEBS 1-TE, a bimodular derivative of DEBS from Saccharopolyspora erythraea, which uses (2S)-methylmalonyl-coenzyme. A to produce both D- and L-methyl centers in its triketide lactone product. We show here that condensation of (2S)-methylmalonyl-CoA in module 2 proceeds with decarboxylative inversion without cleavage of the C-H bond adjacent to the methyl group; in contrast, in module 1 the chain extension process involves loss of the hydrogen attached to C-2 of the methylmalonyl-CoA precursor. The production of the D-methyl center in module 2 without loss of hydrogen from the asymmetric center of the (2S)-methylmalonyl-CoA establishes that condensation takes place with inversion of configuration as in fatty acid biosynthesis. The loss of the key hydrogen from the (2S)-methylmalonyl-CoA to produce the L-methyl center generated in module 1 implies that an additional obligatory epimerization step takes place in that module. The nature and timing of the epimerization remain to be established.

Acyl Coenzyme A↗