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Purification and in vitro reconstitution of the essential protein components of an aromatic polyketide synthase

机译：芳香族聚酮化合物合酶的必需蛋白质成分的纯化和体外重建

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A minimal set of proteins which catalyze the synthesis of aromatic polyketides from malonyl CoA has been purified and partially characterized. Plasmid-encoded actinorhodin (net) ketosynthase/chain-length factor (KS/CLF) complex was purified from Streptomyces coelicolor CH999/pSEK38, and assayed with purified aromatic PKS holo-ACPs which were overproduced and purified from Escherichia coli and phosphopantetheinylated in vitro using purified E. coli holo-ACP synthase, When highly purified preparations of KS/CLF, and holo-ACP failed to catalyze polyketide biosynthesis, a fourth protein was sought and purified from the S. coelicolor CH999 host on the basis of its ability to complement KS, CLF, and holo-ACP in polyketide synthesis. N-terminal sequencing identified this protein as the fatty acid synthase (fabD) malonyl CoA:ACP transacylase (MAT), recruited from primary metabolism. A alpha(2) beta(2) structure was shown for the act KS/CLF complex, and three malonyl-enzyme biosynthetic intermediates were identified, defining an escorted path followed by malonyl groups en route from CoA to polyketide. [References: 26]
机译：纯化和部分表征了催化从丙二酰辅酶A合成芳香族聚酮化合物的最小蛋白质组。从大肠杆菌链霉菌CH999 / pSEK38中纯化质粒编码的放线菌丝蛋白（净）酮合成酶/链长因子（KS / CLF）复合物，并用纯化的芳香族PKS holo-ACPs进行分析，而纯化的芳香族PKS holo-ACP则从大肠杆菌中过量生产和纯化，并在体外使用磷酸泛酰化纯化的大肠杆菌holo-ACP合酶，当高度纯化的KS / CLF制剂和holo-ACP无法催化聚酮化合物的生物合成时，根据其补体能力，从大肠杆菌中提取了第四个蛋白并进行纯化。 KS，CLF和holo-ACP在聚酮化合物的合成中。 N端测序鉴定出该蛋白为脂肪酸合成酶（fabD）丙二酰辅酶A：ACP转酰基酶（MAT），是从一次代谢中募集的。显示了行为KS / CLF复合物的alpha（2）beta（2）结构，并确定了三种丙二酰酶生物合成中间体，定义了从CoA到聚酮化合物的丙二醛基跟随的护送路径。 [参考：26]

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