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首页> 外文期刊>Biochemistry >Mapping the Functional Topology of the Animal Fatty Acid Synthase by Mutant Complementation in Vitro
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Mapping the Functional Topology of the Animal Fatty Acid Synthase by Mutant Complementation in Vitro

机译：通过体外突变互补图绘制动物脂肪酸合酶的功能拓扑

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摘要

An in vitro mutant complementation approach has been used to map the functional topology of the animal fatty acid synthase. A series of knockout mutants was engineered, each mutant compromised in one of the seven functional domains, and heterodimers generated by hybridizing all possible combinations of the mutated subunits were isolated and characterized. Heterodimers comprised of a subunit containing either a #beta#-ketoacyl synthase or malonyl/acetyltransferase mutant, paired with a subunit containing mutations in any one of the other five domains, are active in fatty acid synthesis. Heterodimers in which both subunits carry a knockout mutation in either the dehydrase, enoyl reductase, keto reductase, or acyl carrier protein are inactive. Heterodimers comprised of a subunit containing a thioesterase mutation paired with a subunit containing a mutation in either the dehydrase, enoly reductase, #beta#-ketoacyl reductase, or acyl carrier protein domains exhibit very low fatty acid synthetic ability. The results are consistent with a model for the fatty acid synthase in which the substrate loading and condensation reactions are catalyzed by cooperation of an acyl carrier protein domain of one subunit with the maloyl/acetyltransferase or #beta#-ketoacyl synthase domains, respectively, of either subunit. The #beta#-carbon-processing reactions, responsible for the complete reduction of the #beta#-ketoacyl moiety following each condensation step, are catalyzed by cooperation of an acyl carrier protein domain with the #beta#-ketoacyl reductase, dehydrase, and enoyl reductase domains associated exclusively with the same subunit. The chain-terminating reaction is carried out most efficiently by cooperation of an acyl carrier protein domain with the thioesterase domain of the same subunit. These results are discussed in the context of a revised model for the fatty acid synthase.
机译：已使用体外突变体互补方法来绘制动物脂肪酸合酶的功能拓扑。工程设计了一系列敲除突变体，每个突变体都折入了七个功能域之一，并且分离并表征了通过杂交所有可能的突变亚基组合而产生的异二聚体。异源二聚体由含有＃β＃-酮酰基合酶或丙二酰/乙酰基转移酶突变体的亚基组成，并与其他五个域中任意一个域中含有突变的亚基配对，在脂肪酸合成中具有活性。其中两个亚基均在脱水酶，烯酰还原酶，酮还原酶或酰基载体蛋白中均具有敲除突变的异二聚体是无活性的。由包含硫酯酶突变的亚基与脱水酶，烯醇还原酶，＃β＃-酮酰基还原酶或酰基载体蛋白结构域中的突变的亚基组成的异二聚体表现出非常低的脂肪酸合成能力。该结果与脂肪酸合酶的模型一致，在该模型中，底物负载和缩合反应通过一个亚基的酰基载体蛋白结构域分别与丙二酸的丙二酰/乙酰基转移酶或＃β＃-酮酰基合酶结构域共同催化。任一亚基。负责每个缩合步骤后＃beta＃-酮酰基部分完全还原的＃beta＃-碳加工反应是通过酰基载体蛋白结构域与＃beta＃-酮酰基还原酶，脱水酶和烯酰还原酶结构域仅与同一亚基相关。通过酰基载体蛋白结构域与相同亚基的硫酯酶结构域的协同作用，最有效地进行链终止反应。在脂肪酸合酶的修订模型的背景下讨论了这些结果。

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