Data Science-Driven Analysis of Substrate-Permissive Diketopiperazine Reverse Prenyltransferase NotF: Applications in Protein Engineering and Cascade Biocatalytic Synthesis of (-)-Eurotiumin A.
Data Science-Driven Analysis of Substrate-Permissive Diketopiperazine Reverse Prenyltransferase NotF: Applications in Protein Engineering and Cascade Biocatalytic Synthesis of (-)-Eurotiumin A.
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DOI：
10.1021/jacs.2c06631
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发表时间：
2022-10-26
影响因子：
15
通讯作者：
Sherman, David H.
中科院分区：
文献类型：
--
作者：
Kelly, Samantha P.;V. Shende, Vikram;Flynn, Autumn R.;Dan, Qingyun;Ye, Ying;Smith, Janet L.;Tsukamoto, Sachiko;Sigman, Matthew S.;Sherman, David H.
丙基转移是一种早期的碳氢键(C-H)功能化，广泛存在于一系列具有生物活性的细菌、真菌、植物和后生动物二酮基哌嗪(DKP)生物碱的生物合成中。为了开发一种统一的构建戊烯基化DKP吲哚生物碱的生物催化策略，我们试图鉴定和表征底物允许的C2反向戊烯基转移酶(PT)。作为细胞毒性诺托胺代谢产物生物合成中的第一个剪裁事件，PT NotF催化短链酰胺F的C2反向戊基转移。解析NotF的晶体结构(与天然底物和戊基供体模拟二甲基烯丙基S硫代二磷酸(DMSPP)的络合物)揭示了一个大的暴露于溶剂的活性中心，暗示NotF可能具有非常广泛的底物范围。为了评估NotF的底物选择性，我们合成了一组30个空间和电子分化的色氨基DKPs，其中大多数在合成有用的转化中被NotF选择性地戊烯基化(2到>；99%)。该底物文库的定量表示和描述性统计模型的发展为深入了解NotF底物混杂的分子起源提供了线索。这种方法能够识别控制NotF催化的戊烯转移速率的关键底物描述符(亲电性、大小和灵活性)，并开发出“诱导匹配对接(IFD)指导”的工程策略，提高了我们最大底物的周转率。我们进一步证明了PT NotF与使用黄素单加氧酶BvnB的氧化环化相结合的实用性。这种一锅法体外生物催化级联反应首次实现了海洋真菌天然产物(-)-Eurotiumin A的三步化学酶合成，总收率为60%。
Prenyltransfer is an early-stage carbon–hydrogen bond (C–H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Towards the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF’s substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an “induced fit docking (IFD)-guided” engineering strategy improved turnover of our largest substrates. We further demonstrated the utility of PT NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (–)-eurotiumin A, in three steps and 60% overall yield.
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影响因子：
21.8
作者：
Dan, Qingyun;Newmister, Sean A.;Williams, Robert M.
通讯作者：
Williams, Robert M.
影响因子：
2.9
作者：
Kmunícek, J;Hynková, K;Damborsky, J
通讯作者：
Damborsky, J
影响因子：
15
作者：
Chen, Mengbin;Liu, Chun-Ting;Tang, Yi
通讯作者：
Tang, Yi
影响因子：
2.1
作者：
Greshock, Thomas J.;Grubbs, Alan W.;Williams, Robert M.
通讯作者：
Williams, Robert M.
Notoamides A-D:: 从一种源自海洋的真菌 Aspergillus sp.
影响因子：
16.6
作者：
Kato, Hikaru;Yoshida, Takushi;Tsukamoto, Sachiko
通讯作者：
Tsukamoto, Sachiko