Functional orientation of the acyltransferase domain in a module of the erythromycin polyketide synthase
Functional orientation of the acyltransferase domain in a module of the erythromycin polyketide synthase
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DOI：
10.1021/bi971887n
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发表时间：
1998-02-24
期刊：
影响因子：
2.9
通讯作者：
Khosla, C
中科院分区：
文献类型：
--
作者：
Gokhale, RS;Lau, J;Khosla, C
模块化聚酮酶（PI（Ss），例如6-脱氧聚酮B合酶（DEBS），催化结构复杂且具有药用重要性的天然产物的生物合成。这些大的多酶被组织成一系列的功能单元，称为模块，每个二聚体模块包含两个催化独立的活性位点簇，这些活性位点与脊椎动物脂肪酸脱氢酶的活性位点同源。早期的研究表明，模块由头到尾的同源二聚体组成，其中酮合酶（KS）和酰基载体蛋白（ACP）结构域由相对的亚基贡献，形成催化中心。在这里，我们探测的酰基转移酶（AT）结构域的功能拓扑结构转移到ACP结构域的磷酸泛酰巯基乙胺臂的甲基丙二酰辅酶A的甲基丙二酰部分。使用DEBS的双模块衍生物，通过定点诱变使模块2（AT 2）的AT结构域失活。在体外在失活的AT 2（AT 2度）多肽和失活的KS 1（KS 1度）或KS 2（KS 2度）蛋白之间产生异二聚体蛋白对。这两种杂合蛋白都支持聚酮合成，表明AT 2可以从任一亚基执行其功能。两种杂合蛋白对中的每一种的表观催化速率常数，KS 1度/AT 2度和KS 2度/AT 2度是相同的，表明对于特定的AT 2-ACP 2组合不存在显著的动力学偏好。这些结果表明，AT结构域之间可以共享的两个集群的活性位点在同一个二聚体模块，这种新的结构组织可能提供一个功能优势，有效的生物合成聚酮化合物。
Modular polyketide synthases (PI(Ss),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.