Epothilone biosynthesis: assembly of the methylthiazolylcarboxy starter unit on the EpoB subunit
Epothilone biosynthesis: assembly of the methylthiazolylcarboxy starter unit on the EpoB subunit
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DOI：
10.1016/s1074-5521(01)00064-3
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发表时间：
2001-09-01
影响因子：
--
通讯作者：
Walsh, CT
中科院分区：
文献类型：
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作者：
Chen, HW;O'Connor, S;Walsh, CT
背景：聚酮（PKs）和非核糖体肽（NRP）是由多模块蛋白质组装线通过类似的硫模板介导机制生物合成的具有重要治疗意义的天然产物，称为PK合成酶（PKs）和NRP合成酶（NRPSs）。这两种平行酶系统之间的潜在生产相互作用最近已被证明，发现PK/NRP杂交天然产物可以具有很大的治疗重要性。新发现的一种具有抗肿瘤潜力的药代药PK/NRP产物——纤维素Sorangium epthilone D，作为一种抗肿瘤药物显示出了巨大的潜力。结果：利用多模埃波霉素合成酶两个模块的5个结构域，体外合成了埃波霉素链启动甲基噻唑环作为酰基s酶中间体。从EpoA基因中切除的酰基载体蛋白（ACP）结构域在大肠杆菌中表达，纯化为载子蛋白，然后使用磷酸antetheinyl转移酶Sfp在翻译后引物乙酰辅酶a。四个结构域150 kda的EpoB亚基（环化-腺苷化-氧化酶-肽基载体蛋白结构域：CyA-Ox-PCP）也在大肠杆菌中表达并以可溶性形式纯化。用Sfp和CoASH进行翻译后修饰，将HS-pantP假体基引入载脂蛋白pcp，随后用l -半胱氨酸装载生成Cys-S-PCP酰基酶中间体。当乙酰- s- acp （EpoA）和半胱氨酸- s- EpoB混合时，EpoB的Cy结构域催化乙酰基从EpoA转移到Cys-S-EpoB的氨基上，生成N-Ac-Cys-S-EpoB中间体，该中间体被环化脱水成五元环甲基噻唑啉- s- EpoB。最后，含fmn结构域的EpoB将二氢杂环噻唑啉环氧化成杂芳氧化态，甲基噻唑基羧基- s -EpoB。当其他酰基辅酶a取代基于sfp的载脂蛋白cp结构域的乙酰辅酶a时，产生额外的烷基噻唑基羧基s - epob酰基酶。结论：这些实验建立了跨PKS和NRPS界面的链转移，将乙酰基从EpoA的ACP结构域转移到EpoB，重建了艾替酮合成酶装配线的起点，并在该天然产物链生长过程中将半胱氨酸基团安装并转化为甲基取代杂环。2001爱思唯尔科学有限公司版权所有。
Background: Polyketides (PKs) and non-ribosomal peptides (NRPs) are therapeutically important natural products biosynthesized by multimodular protein assembly lines, termed the PK synthases (PKSs) and NRP synthetases (NRPSs), via a similar thiotemplate-mediated mechanism. The potential for productive interaction between these two parallel enzymatic systems has recently been demonstrated, with the discovery that PK/NRP hybrid natural products can be of great therapeutic importance. One newly discovered PK/NRP product, epothilone D from great potential as an anti-tumor Sorangium cellulosum, has shown great potential as an anti-tumor agent.Results: The chain-initiating methylthiazole ring of epothilone has been generated in vitro as an acyl-S-enzyme intermediate, using five domains from two modules of the polymodular epothilone synthetase. The acyl carrier protein (ACP) domain, excised from the EpoA gene, was expressed in Escherichia coli, purified as an apo protein, and then post-translationally primed with acetyl-CoA using the phosphopantetheinyl transferase enzyme Sfp. The four-domain 150-kDa EpoB subunit (cyclization-adenylation-oxidase-peptidyl carrier protein domains: CyA-Ox-PCP) was also expressed and purified in soluble form from E. coli. Post-translational modification with Sfp and CoASH introduced the HS-pantP prosthetic group to the apo-PCP, enabling subsequent loading with L-cysteine to generate the Cys-S-PCP acyl enzyme intermediate. When acetyl-S-ACP (EpoA) and cysteinyl-S-EpoB were mixed, the Cy domain of EpoB catalyzed acetyl transfer from EpoA to the amino group of the Cys-S-EpoB, generating a transient N-Ac-Cys-S-EpoB intermediate that is cyclized and dehydrated to the five-membered ring methylthiazolinyl-S-EpoB. Finally, the FMN-containing Ox domain of EpoB oxidized the dihydro heterocyclic thiazolinyl ring to the heteroaromatic oxidation state, the methylthiazolylcarboxy-S-EpoB. When other acyl-CoAs were substituted for acetyl-CoA in the Sfp-based priming of the apo-CP domain, additional alkylthiazolylcarboxy-S-EpoB acyl enzymes were produced.Conclusions: These experiments establish chain transfer across a PKS and NRPS interface, Transfer of the acetyl group from the ACP domain of EpoA to EpoB reconstitutes the start of the epothilone synthetase assembly line, and installs and converts a cysteine group into a methyl-substituted heterocycle during this natural product chain growth. (C) 2001 Elsevier Science Ltd. All rights reserved.