Investigation of Substrate Recognition and Biosynthesis in Class IV Lanthipeptide Systems.
Investigation of Substrate Recognition and Biosynthesis in Class IV Lanthipeptide Systems.
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DOI：
10.1021/jacs.8b01323
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发表时间：
2018-05-02
影响因子：
15
通讯作者：
van der Donk WA
中科院分区：
文献类型：
--
作者：
Hegemann JD;van der Donk WA
羊毛硫肽属于核糖体合成和后修饰肽（RIPPs）家族，并被细分为四类。前两类已经被大量研究，但对第III类和第IV类知之甚少。III类和IV类羊毛硫肽合成酶具有相似的蛋白质结构域：N-末端的裂解酶结构域、中心激酶结构域和C-末端环化酶结构域。在这里，我们提供了更深入的了解IV类酶（LanLs）。筛选了一系列推定的生产菌株，以确定四种新的委内瑞拉肽类羊毛硫肽的生产条件，并建立了一个基于大肠杆菌的异源生产系统的第五。后者不仅允许生产完全修饰的核心肽，而且还用作前体肽的突变分析的基础，以鉴定对酶识别重要的区域。这些实验通过体外结合研究进行补充，旨在鉴定LanL酶识别的前导肽区域以及确定酶的哪个结构域识别底物肽。这些研究表明，激酶结构域介导了与前体肽的相互作用，并且前导肽中心至N-末端区域残基的puplex α-螺旋延伸对于酶识别是重要的。此外，体外试验和串联质谱的组合用于阐明这些系统中脱水事件的顺序。
Lanthipeptides belong to the family of ribosomally-synthesized and posttranslationally-modified peptides (RiPPs) and are subdivided into four classes. The first two classes have been heavily studied, but less is known about classes III and IV. The lanthipeptide synthetases of classes III and IV share a similar organization of protein domains: A lyase domain at the N-terminus, a central kinase domain and a C-terminal cyclase domain. Here, we provide deeper insight into class IV enzymes (LanLs). A series of putative producer strains was screened to identify production conditions of four new venezuelin-like lanthipeptides and an Escherichia coli based heterologous production system was established for a fifth. The latter not only allowed production of fully modified core peptide, but was also employed as basis for mutational analysis of the precursor peptide to identify regions important for enzyme recognition. These experiments were complemented by in vitro binding studies aimed at identifying the region of the leader peptide recognized by the LanL enzymes as well as determining which domain of the enzyme is recognizing the substrate peptide. Combined, these studies revealed that the kinase domain is mediating the interaction with the precursor peptide and that a putatively α-helical stretch of residues at the center to N-terminal region of the leader peptide is important for enzyme recognition. In addition, a combination of in vitro assays and tandem mass spectrometry was used to elucidate the order of dehydration events in these systems.
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DOI：
10.1073/pnas.1703663114
发表时间：
2017-09-26
影响因子：
11.1
作者：
Davis, Katherine M.;Schramma, Kelsey R.;Ando, Nozomi
通讯作者：
Ando, Nozomi
拟茎点菌的生物合成研究揭示了子囊菌中核糖体天然产物的广泛途径
DOI：
10.1073/pnas.1522907113
发表时间：
2016-03-29
影响因子：
11.1
作者：
Ding, Wei;Liu, Wan-Qiu;Zhang, Qi
通讯作者：
Zhang, Qi
影响因子：
2.7
作者：
Camarero JA
通讯作者：
Camarero JA
影响因子：
2.1
作者：
Chen, P;Qi, FX;Caufield, PW
通讯作者：
Caufield, PW
影响因子：
14.9
作者：
Chiu, J;March, PE;Tillett, D
通讯作者：
Tillett, D