A tRNA-Dependent Two-Enzyme Pathway for the Generation of Singly and Doubly Methylated Ditryptophan 2,5-Diketopiperazines
A tRNA-Dependent Two-Enzyme Pathway for the Generation of Singly and Doubly Methylated Ditryptophan 2,5-Diketopiperazines
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DOI：
10.1021/bi4004827
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发表时间：
2013-06-18
期刊：
影响因子：
2.9
通讯作者：
Marahiel, Mohamed A.
中科院分区：
文献类型：
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作者：
Giessen, Tobias W.;von Tesmar, Alexander M.;Marahiel, Mohamed A.
大量含有2，5-二酮哌嗪（DKP）部分的生物活性天然产物已从各种微生物来源中分离出来。特别是含葡聚糖的环二肽（CDPs），其结构和功能多样，但其生物合成途径却知之甚少。在这里，我们描述了生物信息学分析的环二肽合酶（CDPS），包含基因簇从放射丝藻跨越2.9 kb，包含两个假定的DKP修饰酶。我们建立了生物合成途径，导致两个甲基化ditryptophan CDPs通过在体内和体外分析。我们的研究鉴定了第一个显示出高底物特异性的CDPS（Amir_4627），其仅合成一种主要产物环（Trp-Trp）（cWW）。它是CDPS家族的第一个成员，可以形成ditryptophan DKP和第一个原核CDPS，其主要产物成分不同于通常在CDPS依赖性CDPs中发现的四种氨基酸（Phe，Leu，Tyr和Met）。我们表明，cWW形成后，S-腺苷-L-甲硫氨酸依赖性N-甲基转移酶（Amir_4628）进行两个连续的甲基化在DKP环氮，并另外表明，它能够甲基化其他四个苯丙氨酸含CDP。这使得Amir_4628成为第一个鉴定的DKP环修饰甲基转移酶。已知作用于含Trp的DKP的大量已知的细菌和真菌来源的修饰酶使得鉴定由小基因编码的用于cWW形成的有效催化剂对于体内组合以及化学酶促方法是有价值的，目的是产生已知CDP天然产物的衍生物或具有潜在改进的或新的生物活性的全新化学实体。
A large number of bioactive natural products containing a 2,5-diketopiperazine (DKP) moiety have been isolated from various microbial sources. Especially tryptophan-containing cyclic dipeptides (CDPs) show great structural and functional diversity, while little is known about their biosynthetic pathways. Here, we describe the bioinformatic analysis of a cyclodipeptide synthase (CDPS)-containing gene cluster from Actinosynnema mirum spanning 2.9 kb that contains two putative DKP-modifying enzymes. We establish the biosynthetic pathway leading to two methylated ditryptophan CDPs through in vivo and in vitro analyses. Our studies identify the first CDPS (Amir_4627) that shows high substrate specificity synthesizing only one main product, cyclo(Trp-Trp) (cWW). It is the first member of the CDPS family that can form ditryptophan DKPs and the first prokaryotic CDPS whose main product constituents differ from the four amino acids (Phe, Leu, Tyr, and Met) usually found in CDPS-dependent CDPs. We show that after cWW formation a S-adenosyl-L-methionine-dependent N-methyltransferase (Amir_4628) conducts two successive methylations at the DKP-ring nitrogens and additionally show that it is able to methylate four other phenylalanine-containing CDPs. This makes Amir_4628 the first identified DKP-ring-modifying methyltransferase. The large number of known modifying enzymes of bacterial and fungal origin known to act upon Trp-containing DKPs makes the identification of a potent catalyst for cWW formation, encoded by a small gene, valuable for combinatorial in vivo as well as chemoenzymatic approaches, with the aim of generating derivatives of known CDP natural products or entirely new chemical entities with potentially improved or new biological activities.