Carreras C W, Khosla C
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Biochemistry. 1998 Feb 24;37(8):2084-8. doi: 10.1021/bi972919+.
A minimal set of proteins which catalyze the synthesis of aromatic polketides from malonyl CoA has been purified and partially characterized. Plasmid-encoded actinorhodin (act) 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 alpha2/beta2 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.
一组催化从丙二酰辅酶A合成芳香族聚酮化合物的最小蛋白质已被纯化并进行了部分表征。从天蓝色链霉菌CH999/pSEK38中纯化出质粒编码的放线紫红素（act）酮合成酶/链长因子（KS/CLF）复合物，并用从大肠杆菌中过量表达并纯化的芳香族聚酮合酶全酰基载体蛋白（holo-ACP）进行测定，这些全酰基载体蛋白在体外使用纯化的大肠杆菌全酰基载体蛋白合酶进行了磷酸泛酰巯基乙胺化。当高度纯化的KS/CLF和全酰基载体蛋白制剂未能催化聚酮化合物生物合成时，基于其在聚酮化合物合成中补充KS、CLF和全酰基载体蛋白的能力，从CH999宿主中寻找并纯化出了第四种蛋白质。N端测序确定该蛋白质为从初级代谢中招募的脂肪酸合酶（fabD）丙二酰辅酶A：酰基载体蛋白转酰基酶（MAT）。act KS/CLF复合物呈现出α2/β2结构，并鉴定出三种丙二酰酶生物合成中间体，确定了丙二酰基团从辅酶A到聚酮化合物的护送路径。
