Zawada R J, Khosla C
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Chem Biol. 1999 Sep;6(9):607-15. doi: 10.1016/s1074-5521(99)80112-4.
Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products. The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase. Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain. We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC).
The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies. Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone. Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate.
Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces. Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS. They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems.
聚酮合酶（PKSs）是细菌的多酶系统，可合成多种天然产物。“最小”PKS由酮合成酶、链长因子、酰基载体蛋白和丙二酰转移酶组成。辅助成分（酮还原酶、芳香化酶和环化酶）参与控制新生聚酮链的氧化水平和环化。我们描述了几种辅助PKS成分的异源表达和重组，包括放线紫红素酮还原酶（act KR）、灰霉素芳香化酶/环化酶（gris ARO/CYC）和四环素霉素芳香化酶/环化酶（tcm ARO/CYC）。
重组的act和tcm PKSs的聚酮产物与先前体内研究中鉴定的产物相同。虽然在最小和辅助PKS成分之间未检测到稳定的蛋白质-蛋白质相互作用，但动力学分析表明，由act最小PKS、act KR和gris ARO/CYC组成的扩展PKS比act最小PKS加act KR或单独的act最小PKS具有更高的周转数。将tcm ARO/CYC添加到tcm最小PKS中也提高了总体速率。
直到最近，PKS亚基功能分析的主要策略还是通过在链霉菌中异源表达重组PKSs。我们的结果证实了一个隐含的假设，即从全细胞系统中分离出的产物是PKS的主要产物。它们还表明，一种中间体在各个亚基之间传递，为这些多酶系统更详细的结构和机制分析铺平了道路。
