Dreier J, Shah A N, Khosla C
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
J Biol Chem. 1999 Aug 27;274(35):25108-12. doi: 10.1074/jbc.274.35.25108.
Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA:ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-beta-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of protein-protein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 +/- 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [(14)C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.
II型聚酮合酶（PKSs）是一类细菌多酶系统，可催化多种天然产物的生物合成。一组核心亚基，由一个酮合成酶、一个链长因子、一个酰基载体蛋白（ACP）以及可能的丙二酰辅酶A：ACP转酰基酶（MAT）组成，形成一个“最小”PKS。它们利用源自丙二酰辅酶A的构件生成特定长度的聚β-酮骨架。在此，我们（a）报告了放线紫红素最小PKS的动力学特性，并且（b）提供了进一步的数据以支持MAT的必要性。动力学分析表明，脱辅基ACP是最小PKS活性的竞争性抑制剂，这证明了ACP的多肽部分与最小PKS其余部分之间蛋白质-蛋白质相互作用的重要性。为进一步支持MAT对PKS活性的必要性，我们展示了两个新发现。首先，我们观察到PKS活性对MAT浓度呈双曲线依赖性，在非常低的量时达到饱和（半最大速率时为19.7±5.1 nM）。由于MAT能够在低于ACP以及酮合成酶/链长因子组分典型浓度1/100的情况下支持PKS活性，因此很难排除PKS反应混合物中存在痕量MAT的可能性。其次，在MAT的亲核活性位点构建了一个S97A突变体。该突变蛋白不仅能够支持PKS活性，还能被[¹⁴C]丙二酰辅酶A共价标记，这表明丝氨酸亲核试剂（在早期研究中是苯甲基磺酰氟抑制的靶点）在II型PKS系统中对于MAT活性是可有可无的。
