Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Am Chem Soc. 2012 May 2;134(17):7359-66. doi: 10.1021/ja2112228. Epub 2012 Apr 23.
Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 Å germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.
灰色链霉菌中的 germicidin 合酶（Gcs）是一种 III 型聚酮合酶（PKS），具有广泛的酰基结合底物灵活性，通过硫酯键与辅酶 A（CoA）或酰基载体蛋白（ACP）相连。通过将 Gcs 与脂肪酸生物合成偶联，在体外重新合成 germicidin。由于 Gcs 具有广泛的底物灵活性，我们直接比较了 Gcs 与酰基-ACP 和酰基-CoA 的动力学特性。Gcs 对酰基-ACP 的催化效率比酰基-CoA 高 10 倍，表明对载体蛋白起始单元转移有强烈的偏好。2.9Å 的 germicidin 合酶晶体结构揭示了典型的 III 型 PKS 结构，以及一个不寻常的、功能未知的螺旋束，似乎扩展了二聚化界面。位于活性位点附近的一对精氨酸残基影响催化活性，但不影响 ACP 结合。这项研究提供了关于 III 型 PKSs 与 ACPs 之间相互作用的新的、令人惊讶的信息，这将有助于构建用于生产新型聚酮的工程化系统。
