Carreras C W, Gehring A M, Walsh C T, Khosla C
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Biochemistry. 1997 Sep 30;36(39):11757-61. doi: 10.1021/bi971350+.
The functional reconstitution of two purified proteins of an aromatic polyketide synthase pathway, the acyl carrier protein (ACP) and holo-ACP synthase (ACPS), is described. Holo-ACPs were enzymatically synthesized from coenzyme A and apo-ACPs using Escherichia coli ACPS. Frenolicin and granaticin holo-ACPs formed in this manner were shown to be fully functional together with the other components of the minimal actinorhodin polyketide synthase (act PKS), resulting in synthesis of the same aromatic polyketides as those formed by the act PKS in vivo. ACPS also catalyzed the transfer of acetyl-, propionyl-, butyryl-, benzoyl-, phenylacetyl-, and malonylphosphopantetheines to apo-ACPs from their corresponding coenzyme As, as detected by electrophoresis and/or mass spectrometry. A steady state kinetic study showed that acetyl-coenzyme A is as efficient an ACPS substrate as coenzyme A, with kcat and Km values of 20 min-1 and 25 microM, respectively. In contrast to acetyl-coenzyme A, enzymatically synthesized acetyl-ACPs were shown to be efficient substrates for the act PKS, indicating that acetyl-ACP is a chemically competent intermediate of aromatic polyketide biosynthesis. Together, these methods provide a valuable tool for dissecting the mechanisms and molecular recognition features of polyketide biosynthesis.
本文描述了芳香族聚酮合酶途径中两种纯化蛋白——酰基载体蛋白（ACP）和全酶 ACP 合酶（ACPS）的功能重建。使用大肠杆菌 ACPS 从辅酶 A 和脱辅基 ACP 酶促合成全酶 ACP。以这种方式形成的弗罗里辛和石榴皮素全酶 ACP 与最小的放线紫红素聚酮合酶（act PKS）的其他组分一起显示出完全功能，从而合成与 act PKS 在体内形成的相同的芳香族聚酮化合物。通过电泳和/或质谱检测，ACPS 还催化乙酰基、丙酰基、丁酰基、苯甲酰基、苯乙酰基和丙二酰磷酸泛酰巯基乙胺从相应的辅酶 A 转移到脱辅基 ACP 上。稳态动力学研究表明，乙酰辅酶 A 作为 ACPS 的底物与辅酶 A 一样有效，其 kcat 和 Km 值分别为 20 min⁻¹ 和 25 μM。与乙酰辅酶 A 相反，酶促合成的乙酰基 ACP 被证明是 act PKS 的有效底物，表明乙酰基 ACP 是芳香族聚酮生物合成的化学活性中间体。总之，这些方法为剖析聚酮生物合成的机制和分子识别特征提供了有价值的工具。
