Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase.
Picromycin synthase (PICS) is a multifunctional, modular polyketide synthase (PKS) that catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide, the macrolide aglycone precursors of the antibiotics picromycin and methymycin, respectively. PICS modules 5 and 6 were each expressed in Escherichia coli with a thioesterase domain at the C-terminus to allow release of polyketide products. The substrate specificity of PICS modules 5+TE and 6+TE was investigated using N-acetylcysteamine thioesters of 2-methyl-3-hydroxy-pentanoic acid as diketide analogues of the natural polyketide chain elongation substrates. PICS module 5+TE could catalyze the chain elongation of only the syn diketide (2S,3R)-4, while PICS module 6+TE processed both syn diastereomers, (2S,3R)-4 and (2R,3S)-5, with a 2.5:1 preference in k(cat)/K(m) for 5 but did not turn over either of the two anti diketides. The observed substrate specificity patterns are in contrast to the 15-100:1 preference for 4 over 5 previously established for several modules of the closely related erythromycin PKS, 6-deoxyerythronolide B synthase (DEBS).
Yin
Yifeng
Y
Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, USA.
Lu
Hongxiang
H
Khosla
Chaitan
C
Cane
David E
DE
D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D018942:Macrolides; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D018942:Macrolides; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C008517:methymycin; C003145:picromycin