title: Erythromycin biosynthesis: kinetic studies on a fully active modular polyketide synthase using natural and unnatural substrates.
abstract: 6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) that catalyzes the biosynthesis of the parent macrolide of erythromycin. On the basis of a recently developed cell-free assay (Pieper et al., 1995a) we report the results of steady-state kinetic studies on a modular PKS. A truncated form of DEBS (DEBS 1+TE), in which DEBS 1 is fused to the thioesterase domain from the C-terminal end of DEBS 3, was used for most of these studies. The overall k(cat) for (2S,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-heptanoic acid delta-lactone (C9-lactone) synthesis is 3.4 min(-1), indicating that the enzyme is at least as active in vitro as in vivo. The apparent K(m) for (2S)-methylmalonyl-CoA consumption by DEBS 1+TE is 24 microM. The catalytic activity of DEBS 1+TE is strongly dependent on the phosphate concentration in the reaction buffer in the range 0-250 mM, suggesting that hydrophobic interactions may be crucial to the assembly of DEBS monomers into a functional complex. Although DEBS 1+TE can convert acetyl-, propionyl-, or butyryl-CoA into the corresponding C8-, C9-, and C10-lactones (Pieper et al., 1995b), it has a 32-fold preference for a propionate primer over an acetate primer and a 7.5-fold preference for a propionate primer over a butyrate primer. In the absence of any added primer unit, synthesis can be primed via decarboxylation of methylmalonyl-CoA; under these conditions the overall k(cat) for polyketide synthesis remains unchanged. Decarboxylation of methylmalonyl-CoA is negligible in the presence of saturating concentrations of propionyl-CoA but competes with the priming of the enzyme by acetyl-CoA or butyryl-CoA. The k(cat) for 6-deoxyerythronolide B synthesis by the complete DEBS is 0.5 min(-1). Under these assay conditions, the C9-lactone is also produced as an abortive chain elongation product with a k(cat) of 0.23 min(-1), presumably due to inefficient assembly of the multimeric protein complex involving DEBS 1, 2, and 3. Together, these results provide the first comprehensive kinetic insights into a fully active modular PKS.
authors.affiliation: Department of Chemical Engineering, Stanford University, California 94305-5025, USA.
authors.forename: R
authors.initials: R
authors.lastname: Pieper
authors.forename: S
authors.initials: S
authors.lastname: Ebert-Khosla
authors.forename: D
authors.initials: D
authors.lastname: Cane
authors.forename: C
authors.initials: C
authors.lastname: Khosla
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism; D004917:Erythromycin / Q000096:biosynthesis*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D013302:Streptomyces / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D002264:Carboxylic Acids; D009097:Multienzyme Complexes; D010710:Phosphates; C015357:methylmalonyl-coenzyme A; D009249:NADP; D004917:Erythromycin