Purification and Characterization of Bimodular and Trimodular Derivatives of the Erythromycin Polyketide Synthase

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• 作者：Rembert Pieper ; Rajesh S. Gokhale ; Guanglin Luo ; David E. Cane ; and Chaitan Khosla
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：February 18, 1997
• 年：1997
• 卷：36
• 期：7
• 页码：1846 - 1851
• 全文大小：246K
• 年卷期：v.36,no.7(February 18, 1997)
• ISSN：1520-4995

文摘

Modular polyketide synthases (PKSs), such as the6-deoxyerythronolide B synthase (DEBS),catalyze the biosynthesis of structurally complex and medicinallyimportant natural products. DEBS isa dimeric protein complex that consists of three large multidomainpolypeptide chains, DEBS 1, DEBS2, and DEBS 3. In turn, each polypeptide includes two modules,where one module is responsible for asingle round of condensation and associated reduction reactions. Ahybrid protein comprised of the firsttwo modules of DEBS fused to a thioesterase domain (DEBS 1+TE) waspurified to homogeneity in afully active form (k_cat = 4.8 min^-1). Synthesis of the anticipated triketide lactonerequired the presenceof (2RS)-methylmalonyl-CoA and NADPH. When available,propionyl-CoA is the preferred source ofprimer units. However, in its absence the enzyme can derive primerunits via decarboxylation of amethylmalonyl extender. The two subunits of an engineeredtrimodular derivative of DEBS, DEBS 1and module 3 of DEBS 2 linked to the TE domain (module 3+TE), werealso individually purified andreconstituted to produce the expected tetraketide lactone invitro (k_cat = 0.23 min^-1).The considerablylower specific activity of this trimodular PKS relative to itsbimodular counterpart presumably reflectsinefficient association between DEBS 1 and module 3+TE. Asexpected, module 3+TE could be efficientlycross-linked as a homodimer. In contrast, no cross-links weredetectable between modules 2 and 3, eventhough biosynthesis of the tetraketide requires transient interactionsto occur between these two modules.Since module 3 only contains the minimal set of active sitesrequired in a module (a ketosynthase, anacyltransferase, and an acyl carrier protein domain) and is the firstactive unimodular protein to be purifiedto homogeneity, it represents an attractive target for futurebiophysical and structural studies.