Kim Chu-Young, Alekseyev Viktor Y, Chen Alice Y, Tang Yinyan, Cane David E, Khosla Chaitan
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Biochemistry. 2004 Nov 9;43(44):13892-8. doi: 10.1021/bi048418n.
The hallmark of a type I polyketide synthase (PKS), such as the 6-deoxyerythronolide B synthase (DEBS), is the presence of catalytic modules comprised of covalently fused domains acting together to catalyze one round of chain elongation. In addition to an obligate ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), a module may also include a ketoreductase (KR), dehydratase (DH), and/or enoyl reductase (ER) domain. The size, flexibility, and fixed domain-domain stoichiometry of these PKS modules present challenges for structural, mechanistic, and protein-engineering studies. Here, we have harnessed the power of limited proteolysis and heterologous protein expression to isolate and characterize individual domains of module 3 of DEBS, a 150-kD protein consisting of a KS, an AT, an ACP, and an inactive KR domain. Two interdomain boundaries were identified via limited proteolysis, which led to the production of a 90-kD KS-AT, a 142-kD KS-AT-KR(0), and a 10-kD ACP as structurally stable stand-alone proteins. Each protein was shown to possess the requisite catalytic properties. In the presence of the ACP, both the KS-AT and the KS-AT-KR(0) proteins were able to catalyze chain elongation as well as the intact parent module. Separation of the KS from the ACP enabled direct interrogation of the KS specificity for both the nucleophilic substrate and the partner ACP. Malonyl and methylmalonyl extender units were found to be equivalent substrates for chain elongation. Whereas ACP2 and ACP4 of DEBS could be exchanged for ACP3, ACP6 was a substantially poorer partner for the KS. Remarkably, the newly identified proteolytic sites were conserved in many PKS modules, raising the prospect of developing improved methods for the construction of hybrid PKS modules by engineering domain fusions at these interdomain junctions.
I型聚酮合酶（PKS）的标志，如6-脱氧红霉内酯B合酶（DEBS），是存在由共价融合结构域组成的催化模块，这些结构域共同作用以催化一轮链延伸。除了必需的酮缩合酶（KS）、酰基转移酶（AT）和酰基载体蛋白（ACP）外，一个模块还可能包括酮还原酶（KR）、脱水酶（DH）和/或烯酰还原酶（ER）结构域。这些PKS模块的大小、灵活性和固定的结构域-结构域化学计量比给结构、机制和蛋白质工程研究带来了挑战。在这里，我们利用有限蛋白酶解和异源蛋白表达的力量，分离并表征了DEBS模块3的各个结构域，DEBS是一种150-kD的蛋白质，由KS、AT、ACP和一个无活性的KR结构域组成。通过有限蛋白酶解确定了两个结构域间的边界，这导致产生了作为结构稳定的独立蛋白的90-kD KS-AT、142-kD KS-AT-KR(0)和10-kD ACP。每个蛋白都显示具有必需的催化特性。在ACP存在的情况下，KS-AT和KS-AT-KR(0)蛋白都能够催化链延伸，与完整的亲本模块一样。KS与ACP的分离使得能够直接探究KS对亲核底物和伴侣ACP的特异性。发现丙二酰和甲基丙二酰延伸单元是链延伸的等效底物。虽然DEBS的ACP2和ACP4可以被ACP3替换，但ACP6对于KS来说是一个相当差的伴侣。值得注意的是，新确定的蛋白水解位点在许多PKS模块中是保守的，这为通过在这些结构域间连接处工程化结构域融合来开发改进的构建杂合PKS模块的方法带来了前景。
