Interrogation of global active site occupancy of a fungal iterative polyketide synthase reveals strategies for maintaining biosynthetic fidelity.
Interrogation of global active site occupancy of a fungal iterative polyketide synthase reveals strategies for maintaining biosynthetic fidelity.
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DOI：
10.1021/ja3016389
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发表时间：
2012-04-18
影响因子：
15
通讯作者：
Townsend, Craig A.
中科院分区：
文献类型：
--
作者：
Vagstad, Anna L.;Bumpus, Stefanie B.;Belecki, Katherine;Kelleher, Neil L.;Townsend, Craig A.
非还原性迭代聚酮酶（NR-PKS）是真菌芳香族天然产物的核心组装酶。尽管最近在该领域取得了进展，但这些大合成酶组装聚酮化合物的许多机制细节仍然未知。为了扩大我们对底物装载，聚酮延长，环化和产物释放，活性位点占用和产物输出的理解，通过傅立叶变换质谱法使用去甲甲嘌呤酸蒽酮生产聚酮合酶，PksA，从黄曲霉毒素生物合成途径寄生曲霉进行了探索。在这里，我们报告的同时观察共价中间体的所有催化结构域的PksA在体外重建反应。这些数据提供了迭代催化的快照，并揭示了C-末端硫酯酶结构域在Claisen/Dieckmann环化和产物释放中最近建立的合成作用之外的未被充分认识的编辑功能。硫酯酶催化水解的特异性进行了探索，使用生物合成相关的蛋白质结合和小分子酰基底物，并证明对己酰基和乙酰基的活性，但不丙二酰基。聚酮化合物的延伸的加工能力的支持的nonhydrolyzable丙二酰类似物，以捕获中间链长度的产品，并通过检测观察到的共价结合，只有完全扩展的物种，并作为主要产品释放，PksA的能力。与起始单元转移相比，丙二酰转移酶结构域的高占有率和丙二酰转移的快速相对速率表明，将延伸单元快速加载到载体结构域上有利于以动力学上有利于最终产物形成的方式进行有效的链延伸。
Nonreducing iterative polyketide synthases (NR-PKSs) are responsible for assembling the core of fungal aromatic natural products with diverse biological properties. Despite recent advances in the field, many mechanistic details of polyketide assembly by these megasynthases remain unknown. To expand our understanding of substrate loading, polyketide elongation, cyclization, and product release, active site occupancy and product output were explored by Fourier transform mass spectrometry using the norsolorinic acid anthrone-producing polyketide synthase, PksA, from the aflatoxin biosynthetic pathway in Aspergillus parasiticus. Here we report the simultaneous observation of covalent intermediates from all catalytic domains of PksA from in vitro reconstitution reactions. The data provide snapshots of iterative catalysis and reveal an underappreciated editing function for the C-terminal thioesterase domain beyond its recently established synthetic role in Claisen/Dieckmann cyclization and product release. The specificity of thioesterase catalyzed hydrolysis was explored using biosynthetically relevant protein-bound and small molecule acyl substrates, and demonstrated activity against hexanoyl and acetyl, but not malonyl. Processivity of polyketide extension was supported by the inability of a nonhydrolyzable malonyl analog to trap products of intermediate chain lengths and by the detection of only fully extended species observed covalently bound to, and as the predominant products released by, PksA. High occupancy of the malonyl transacylase domain and fast relative rate of malonyl transfer compared to starter unit transfer indicate that rapid loading of extension units onto the carrier domain facilitates efficient chain extension in a manner kinetically favorable to ultimate product formation.
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DOI：
10.1038/nrmicro2465
发表时间：
2010-12
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
通讯作者：
--
DOI：
10.1073/pnas.0913531107
发表时间：
2010-04-06
影响因子：
11.1
作者：
Korman, Tyler Paz;Crawford, Jason M.;Tsai, Shiou-Chuan
通讯作者：
Tsai, Shiou-Chuan
影响因子：
5.7
作者：
Bruner, SD;Weber, T;Stubbs, MT
通讯作者：
Stubbs, MT
影响因子：
15
作者：
FU, H;EBERTKHOSLA, S;KHOSLA, C
通讯作者：
KHOSLA, C
影响因子：
1.1
作者：
BROBST, SW;TOWNSEND, CA
通讯作者：
TOWNSEND, CA