Ketosynthases in the Initiation and Elongation Modules of Aromatic Polyketide Synthases Have Orthogonal Acyl Carrier Protein Specificity

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• 作者：Yi Tang ; Taek Soon Lee ; Seiji Kobayashi ; and Chaitan Khosla
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：June 3, 2003
• 年：2003
• 卷：42
• 期：21
• 页码：6588 - 6595
• 全文大小：208K
• 年卷期：v.42,no.21(June 3, 2003)
• ISSN：1520-4995

文摘

Many bacterial aromatic polyketides are synthesized by type II polyketide synthases (PKSs)which minimally consist of a ketosynthase-chain length factor (KS-CLF) heterodimer, an acyl carrierprotein (ACP), and a malonyl-CoA:ACP transacylase (MAT). This minimal PKS initiates polyketidebiosynthesis by decarboxylation of malonyl-ACP, which is catalyzed by the KS-CLF complex and leadsto incorporation of an acetate starter unit. In non-acetate-primed PKSs, such as the frenolicin (fren) PKSand the R1128 PKS, decarboxylative priming is suppressed in favor of chain initiation with alternativeacyl groups. Elucidation of these unusual priming pathways could lead to the engineered biosynthesis ofpolyketides containing novel starter units. Unique to some non-acetate-primed PKSs is a second catalyticmodule comprised of a dedicated homodimeric KS, an additional ACP, and a MAT. This initiation moduleis responsible for starter-unit selection and catalysis of the first chain elongation step. To elucidate theprotein-protein recognition features of this dissociated multimodular PKS system, we expressed andpurified two priming and two elongation KSs, a set of six ACPs from diverse sources, and a MAT. In thepresence of the MAT, each ACP was labeled with malonyl-CoA rapidly. In the presence of a KS-CLFand MAT, all ACPs from minimal PKSs supported polyketide synthesis at comparable rates (k_cat between0.17 and 0.37 min^-1), whereas PKS activity was attenuated by at least 50-fold in the presence of an ACPfrom an initiation module. In contrast, the opposite specificity pattern was observed with priming KSs:while ACPs from initiation modules were good substrates, ACPs from minimal PKSs were significantlypoorer substrates. Our results show that KS-CLF and KSIII recognize orthogonal sets of ACPs, and theadditional ACP is indispensable for the incorporation of non-acetate primer units. Sequence alignmentsof the two classes of ACPs identified a tyrosine residue that is unique to priming ACPs. Site-directedmutagenesis of this amino acid in the initiation and elongation module ACPs of the R1128 PKS confirmedthe importance of this residue in modulating interactions between KSs and ACPs. Our study providesnew biochemical insights into unusual chain initiation mechanisms of bacterial aromatic PKSs.