Tolerance and specificity of recombinant 6-methylsalicyclic acid synthase.
Tolerance and specificity of recombinant 6-methylsalicyclic acid synthase.
复制标题
重组 6-甲基水杨酸合酶的耐受性和特异性。
DOI：
10.1006/mben.1999.0113
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发表时间：
1999
影响因子：
8.4
通讯作者：
Khosla,C
中科院分区：
文献类型：
--
作者：
Richardson,MT;Pohl,NL;Kealey,JT;Khosla,C
6-甲基水杨酸合酶（6-Methylsalicylic acid synthase，MSAS）是一种来源于扩展青霉（Penicillium patulum）的真菌聚酮合酶，它可能是最简单的聚酮合酶，具有多功能结构域、对乙酰辅酶A和丙二酰辅酶A底物高度特异性、链长控制和区域特异性酮还原等特点。最近，MSAS在大肠杆菌和酿酒酵母中得到了功能性表达，导致了6-甲基水杨酸在这些宿主中的工程生物合成。这些发展已经为详细的机制研究这个模型systems.ResultsA三步纯化程序开发，以获得>95%纯度的MSAS从提取物的E。杆菌如前所述的酶分离自P。在patiulum中，重组酶在乙酰辅酶A、丙二酰辅酶A和NADPH的存在下产生6-甲基水杨酸（还原的四酮化合物），但在不存在NADPH的情况下产生三乙酸内酯（未还原的三酮化合物）。与此观察结果一致，在酮还原酶结构域的高度保守的核苷酸结合基序的点突变也导致在体内产生三乙酸内酯。该酶对非天然引物单位（包括丙酰-和丁酰-CoA）表现出一定的耐受性，但不能掺入来自（R，S）-甲基丙二酰-CoA的延伸物单位。有趣的是，MSAS很容易接受theNacetylcysteamine（NAC）类似物丙二酰辅酶Aas substrate.ConclusionsNAC硫酯是简单的，成本效益的类似物CoA硫酯底物，因此提供了一个简便的策略，用于探测的分子识别功能的聚酮酶使用非天然的积木。两种酶均能产生4-羟基-6-甲基-2-吡喃酮。大肠杆菌和酵母说明了这些宿主的代谢工程以产生非天然聚酮化合物的可行性。最后，这里描述的重组MSAS的丰富来源提供了一个机会，研究这个迷人的模型系统使用的结构，机械和诱变方法的组合。
Background6-Methylsalicylic acid synthase (MSAS), a fungal polyketide synthase fromPenicillium patulum, is perhaps the simplest polyketide synthase that embodies several hallmarks of this family of multifunctional enzymes—a large multidomain protein, a high degree of specificity toward acetyl-CoA and malonyl-CoA substrates, chain length control, and regiospecific ketoreduction. MSAS has recently been functionally expressed inEscherichia coliandSaccharomyces cerevisiae, leading to the engineered biosynthesis of 6-methylsalicylic acid in these hosts. These developments have set the stage for detailed mechanistic studies of this model system.ResultsA three--step purification procedure was developed to obtain >95% pure MSAS from extracts ofE. coli. As reported earlier for the enzyme isolated fromP. patulum, the recombinant enzyme produced 6-methylsalicylic acid (a reduced tetraketide) in the presence of acetyl-CoA, malonyl-CoA, and NADPH, but triacetic acid lactone (an unreduced triketide) in the absence of NADPH. Consistent with this observation, point mutations in the highly conserved nucleotide-binding motif of the ketoreductase domain also led to production of triacetic acid lactone in vivo. The enzyme showed some tolerance toward nonnatural primer units including propionyl- and butyryl-CoA, but was incapable of incorporating extender units from (R, S)-methylmalonyl-CoA. Interestingly, MSAS readily accepted theN-acetylcysteamine (NAC) analog of malonyl-CoA as a substrate.ConclusionsNAC thioesters are simple, cost-effective analogs of CoA thioester substrates, and therefore provide a facile strategy for probing the molecular recognition features of polyketide synthases using unnatural building blocks. The ability to produce 4-hydroxy-6-methyl-2-pyrone in bothE. coliand yeast illustrates the feasibility of metabolic engineering of these hosts to produce unnatural polyketides. Finally, the abundant source of recombinant MSAS described here provides an opportunity to study this fascinating model system using a combination of structural, mechanistic, and mutagenesis approaches.