Gokhale R S, Lau J, Cane D E, Khosla C
Department of Chemical Engineering, Chemistry, and Biochemistry, Stanford University, Stanford California 94305-5025, USA.
Biochemistry. 1998 Feb 24;37(8):2524-8. doi: 10.1021/bi971887n.
Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multienzymes are organized into a series of functional units known as modules. Each dimeric module contains two catalytically independent clusters of active sites homologous to those of vertebrate fatty acid synthases. Earlier studies have shown that modules consist of head-to-tail homodimers in which ketosynthase (KS) and acyl carrier protein (ACP) domains are contributed by opposite subunits to form a catalytic center. Here, we probe the functional topology of the acyltransferase (AT) domain which transfers the methylmalonyl moiety of methylmalonyl-CoA onto the phosphopantetheine arm of the ACP domain. Using a bimodular derivative of DEBS, the AT domain of module 2 (AT2) was inactivated by site-directed mutagenesis. Heterodimeric protein pairs were generated in vitro between the inactivated AT2 (AT2 degrees) polypeptide and an inactive KS1 (KS1 degrees) or KS2 (KS2 degrees) protein. Both of these hybrid proteins supported polyketide synthesis, suggesting that AT2 can perform its function from either subunit. The apparent catalytic rate constants for each of the two hybrid protein pairs, KS1 degrees/AT2 degrees and KS2 degrees/AT2 degrees, were identical, indicating that no significant kinetic preference exists for a particular AT2-ACP2 combination. These results suggest that the AT domain can be shared between the two clusters of active sites within the same dimeric module. Such a novel structural organization might provide a functional advantage for the efficient biosynthesis of polyketides.
模块化聚酮合酶（PKSs），如6-脱氧红霉内酯B合酶（DEBS），催化结构复杂且具有重要药用价值的天然产物的生物合成。这些大型多酶由一系列称为模块的功能单元组成。每个二聚体模块包含两个与脊椎动物脂肪酸合酶的活性位点同源的催化独立簇。早期研究表明，模块由头对头同型二聚体组成，其中酮合成酶（KS）和酰基载体蛋白（ACP）结构域由相对的亚基贡献以形成催化中心。在这里，我们探究了将甲基丙二酰辅酶A的甲基丙二酰部分转移到ACP结构域的磷酸泛酰巯基乙胺臂上的酰基转移酶（AT）结构域的功能拓扑结构。使用DEBS的双模块衍生物，通过定点诱变使模块2的AT结构域（AT2）失活。在体外，在失活的AT2（AT2°）多肽与无活性的KS1（KS1°）或KS2（KS2°）蛋白之间产生异源二聚体蛋白对。这两种杂合蛋白都支持聚酮化合物的合成，表明AT2可以从任何一个亚基发挥其功能。两个杂合蛋白对KS1°/AT2°和KS2°/AT2°各自的表观催化速率常数相同，表明对于特定的AT2-ACP2组合不存在明显的动力学偏好。这些结果表明，AT结构域可以在同一二聚体模块内的两个活性位点簇之间共享。这种新颖的结构组织可能为聚酮化合物的高效生物合成提供功能优势。
