Park Hyunjun, Kevany Brian M, Dyer David H, Thomas Michael G, Forest Katrina T
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
PLoS One. 2014 Oct 23;9(10):e110965. doi: 10.1371/journal.pone.0110965. eCollection 2014.
We have previously shown that the acyl transferase domain of ZmaA (ZmaA-AT) is involved in the biosynthesis of the aminopolyol polyketide/nonribosomal peptide hybrid molecule zwittermicin A from cereus UW85, and that it specifically recognizes the precursor hydroxymalonyl-acyl carrier protein (ACP) and transfers the hydroxymalonyl extender unit to a downstream second ACP via a transacylated AT domain intermediate. We now present the X-ray crystal structure of ZmaA-AT at a resolution of 1.7 Å. The structure shows a patch of solvent-exposed hydrophobic residues in the area where the AT is proposed to interact with the precursor ACP. We addressed the significance of the AT/ACP interaction in precursor specificity of the AT by testing whether malonyl- or methylmalonyl-ACP can be recognized by ZmaA-AT. We found that the ACP itself biases extender unit selection. Until now, structural information for ATs has been limited to ATs specific for the CoA-linked precursors malonyl-CoA and (2S)-methylmalonyl-CoA. This work contributes to polyketide synthase engineering efforts by expanding our knowledge of AT/substrate interactions with the structure of an AT domain that recognizes an ACP-linked substrate, the rare hydroxymalonate. Our structure suggests a model in which ACP interaction with a hydrophobic motif promotes secondary structure formation at the binding site, and opening of the adjacent substrate pocket lid to allow extender unit binding in the AT active site.
我们之前已经表明，蜡状芽孢杆菌UW85的氨基多元醇聚酮/非核糖体肽杂合分子两性霉素A的生物合成涉及ZmaA的酰基转移酶结构域（ZmaA-AT），并且它特异性识别前体羟基丙二酰-酰基载体蛋白（ACP），并通过转酰化的AT结构域中间体将羟基丙二酰延伸单元转移到下游的第二个ACP上。我们现在展示了分辨率为1.7 Å的ZmaA-AT的X射线晶体结构。该结构显示在AT与前体ACP相互作用的区域有一片暴露于溶剂的疏水残基区域。我们通过测试ZmaA-AT是否能识别丙二酰-或甲基丙二酰-ACP，探讨了AT/ACP相互作用在AT前体特异性中的重要性。我们发现ACP本身会影响延伸单元的选择。到目前为止，酰基转移酶的结构信息仅限于对与辅酶A连接的前体丙二酰辅酶A和（2S）-甲基丙二酰辅酶A具有特异性的酰基转移酶。这项工作通过扩展我们对识别与ACP连接的底物（罕见的羟基丙二酸）的AT结构域的AT/底物相互作用的了解，为聚酮合酶工程研究做出了贡献。我们的结构提出了一个模型，其中ACP与疏水基序的相互作用促进了结合位点二级结构的形成，并打开了相邻底物口袋的盖子，以便延伸单元在AT活性位点结合。
