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首页> 外文期刊>Biochemistry >Utilization of enzymatically phosphopantetheinylated acyl carrier proteins and acetyl-acyl carrier proteins by the actinorhodin polyketide synthase
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Utilization of enzymatically phosphopantetheinylated acyl carrier proteins and acetyl-acyl carrier proteins by the actinorhodin polyketide synthase

机译:肌动球蛋白聚酮化合物合酶对酶促磷酸泛素化酰基载体蛋白和乙酰基酰基载体蛋白的利用

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摘要

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.
机译:描述了芳香族聚酮化合物合酶途径的两个纯化蛋白,酰基载体蛋白(ACP)和全ACP合酶(ACPS)的功能重构。使用大肠杆菌ACPS从辅酶A和载脂蛋白ACP酶促合成Holo-ACP。已显示以这种方式形成的Frenolicin和Granatinin完整ACP与最小肌动蛋白丁酮多酮合酶(act PKS)的其他组分一起具有完全的功能,从而合成了与Act PKS在体内形成的芳香族聚酮相同的芳香族聚酮。如通过电泳和/或质谱检测,ACPS还催化了乙酰基,丙酰基,丁酰基,苯甲酰基,苯乙酰基和丙二酰基磷酸泛素从其相应的辅酶A向脱辅基-ACP的转移。稳态动力学研究表明,乙酰辅酶A与辅酶A一样,是一种有效的ACPS底物,其kcat和Km值分别为20 min-1和25 microM。与乙酰辅酶A相反,酶促合成的乙酰基ACP被证明是有效的PKS底物,表明乙酰基ACP是芳香族聚酮化合物生物合成的化学活性中间体。总之,这些方法为剖析聚酮化合物生物合成的机理和分子识别特征提供了有价值的工具。

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