Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis.
Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis.
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DOI：
10.1021/jacs.6b09748
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发表时间：
2016-12-14
影响因子：
15
通讯作者：
Aldrich CC
中科院分区：
文献类型：
--
作者：
Fiers WD;Dodge GJ;Sherman DH;Smith JL;Aldrich CC
聚酮合成酶(PKS)作为合成生物学平台在生产新型治疗剂、生物燃料和商品化学品方面仍有很大的应用前景。脱水酶催化结构域在聚酮生物合成过程中起着重要的作用，它通过使新生聚酮中间体脱水来提供烯烃。我们对控制底物特异性和选择性的DH域的详细机制和结构基础的了解仍然有限，从而阻碍了我们合理地重新设计PKS的努力。库拉酸途径含有罕见的多种可能的双键排列，包含共轭烯烃以及顺式和反式烯烃，为聚酮脱水提供了一个无与伦比的模型系统。利用合成的底物对与咖喱酸合成有关的四个水解域进行了体外表征，并用LC-MS/MS分析了其活性。这些研究得到了全反式形成三烯酸的Curk脱水酶的完整的动力学特征，其kcat为72 S−1，比已报道的任何PKS水解区大三个数量级以上。基于与截断底物的孵育研究，为CurJ和CurH脱水酶提出了一种新的立体专一性的二烯形成机制，该机制涉及乙烯基烯醇酸中间体。为了阐明底物与酶的相互作用，将合成的底物与CurJ-Dh的His-Asp催化二聚体中的非活性Phe取代反应共结晶。由此得到的络合物表明了二烯酸酯形成的结构基础，并首次揭示了聚酮类天然产物中形成烯烃所必需的酶-底物相互作用。这种对正则和非正则脱水机制的研究揭示了一些水解域固有的隐藏的催化活性，这些活性可能被用于合成生物学的未来应用。
Polyketide synthase (PKS) enzymes continue to hold great promise as synthetic biology platforms for the production of novel therapeutic agents, biofuels and commodity chemicals. Dehydratase (DH) catalytic domains play an important role during polyketide biosynthesis through the dehydration of the nascent polyketide intermediate to provide olefins. Our understanding of the detailed mechanistic and structural underpinning of DH domains that control substrate specificity and selectivity remains limited, thus hindering our efforts to rationally re-engineer PKSs. The curacin pathway houses a rare plurality of possible double bond permutations containing conjugated olefins as well as both cis- and trans-olefins, providing an unrivaled model system for polyketide dehydration. All four DH domains implicated in curacin biosynthesis were characterized in vitro using synthetic substrates and activity was measured by LC-MS/MS analysis. These studies resulted in complete kinetic characterization of the all trans trienoate-forming CurK dehydratase, whose kcat of 72 s−1 is more than three-orders of magnitude greater than any previously reported PKS DH domain. A novel stereospecific mechanism for diene formation involving a vinylogous enolate intermediate is proposed for the CurJ and CurH dehydratases based on incubation studies with truncated substrates. A synthetic substrate was co-crystallized with a catalytically inactive Phe substitution in the His-Asp catalytic dyad of CurJ DH to elucidate substrate-enzyme interactions. The resulting complex suggested the structural basis for dienoate formation and provided the first glimpse into the enzyme-substrate interactions essential for the formation of olefins in polyketide natural products. This examination of both canonical and non-canonical dehydration mechanisms reveals hidden catalytic activity inherent in some DH domains that may be leveraged for future applications in synthetic biology.
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影响因子：
8.4
作者：
Fiers WD;Dodge GJ;Li Y;Smith JL;Fecik RA;Aldrich CC
通讯作者：
Aldrich CC
DOI：
10.1016/j.str.2009.10.018
发表时间：
2010-01-13
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Akey DL;Razelun JR;Tehranisa J;Sherman DH;Gerwick WH;Smith JL
通讯作者：
Smith JL
DOI：
10.1002/anie.201005280
发表时间：
2011-03-14
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Gu L;Eisman EB;Dutta S;Franzmann TM;Walter S;Gerwick WH;Skiniotis G;Sherman DH
通讯作者：
Sherman DH
影响因子：
4.8
作者：
Gaitatzis, N;Silakowski, B;Müller, R
通讯作者：
Müller, R
影响因子：
3.2
作者：
Caffrey, P
通讯作者：
Caffrey, P