Biosynthesis of the α-nitro-containing cyclic tripeptide psychrophilin.
Biosynthesis of the α-nitro-containing cyclic tripeptide psychrophilin.
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DOI：
10.1038/ja.2016.33
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发表时间：
2016-07
期刊：
影响因子：
--
通讯作者：
Tang Y
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文献类型：
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作者：
Zhao M;Lin HC;Tang Y
嗜冷蛋白A-H（1-8）是从不同青霉属物种分离的环状三肽（图1）。1-4这些天然产物通过非核糖体肽合成酶（NRPS）的作用由L-色氨酸、非天然氨基酸邻氨基苯甲酸酯（Ant）和另外的天然L-氨基酸（脯氨酸：1，5-8;缬氨酸：2;丙氨酸：3;和亮氨酸：4）中的每一个分子合成。例如，预期嗜冷蛋白B 2从连接到相应NRPS的最后一个硫醇化结构域的L-Trp-L-Val-Ant三肽基硫酯环化。近年来，含蚂蚁的真菌肽基生物碱已被充分表征，其中大部分是结构复杂的多环化合物。5然而，嗜冷蛋白的13元大环的产生是非常不寻常的，突出表现为Ant的羧酸酯和L-Trp的吲哚氮之间形成酰胺键，而不是预期的L-Trp的α-NH 2。有趣的是，在1-4中，L-Trp的α-NH 2被氧化成很少观察到的硝基; 1-3而在5-8中，相同的氨基被乙酰化并进一步甲基化。4因此，L-Trp α-NH 2修饰（氧化或乙酰化/甲基化）相对于大环化的时间是一个有趣的生物合成问题（图2）。与吲哚氮相比，α-NH 2基团在大环化反应中的反应性显著更高。通过α-NH 2的亲核环化（路径c）是典型的NRPS大环化反应，并将导致形成10元大环内酰胺11，该大环内酰胺11可能发生自发喹唑啉形成（图2），如在其他含Ant和L-Trp的三肽真菌NRPs（如富米喹唑啉F）中观察到的。6，7因此，我们假设L-Trp的α-氨基可能在环化之前被氧化成硝基，以防止形成11，并允许吲哚氮作为亲核试剂（路径B）。或者，环化反应中吲哚氮的参与可以完全由NRPS的环化结构域控制，并且在形成环状三肽9之后发生α-NH 2的氧化（路径a）。为了研究2和3的生物合成，我们使用Illumina HiSeq对产生耐冷真菌Penicillium rivulum（IBT 24420）的基因组进行测序。1组装~ 36 Mb的基因组，并使用AntiSMASH扫描NRPS基因。8共发现30个编码NRPS的基因，大小在单模块和六模块之间。使用针对激活Ant的腺苷酸化结构域确定的氨基酸特异性代码，发现了9个不同的模块，并进一步分析了潜在的psy簇。发现在支架46和支架182中编码的两个候选簇是最可能的。支架182中编码的簇含有三模块NRPS（Pv 182 -1）和细胞色素P450单加氧酶（P450，Pv 182 -2）。虽然NRPS和P450是预期产生2和3所需的最低限度的酶，但发现Ant特异性A结构域位于NRPS的第一个模块中，因此与氨基酸活化的预测顺序不一致（1：L-Trp; 2：L-Val/L-Ala; 3：Ant）。事实上，该簇的遗传失活不会导致嗜冷蛋白产生的消除（补充图S6）。发现两个独立的NRPS编码基因聚集在支架46中（图2和补充表S2），包括具有A-PCP-CA-PCP-C结构域组织的双模块NRPS Pv 46 -49（结构域缩写：A：腺苷酸化; PCP：肽基载体蛋白; C：缩合）和具有CA-PCP-CT结构域组织的单模块NRPS Pv 46 -51。
Psychrophilins A–H (1–8) are cyclic tripeptides isolated from different Penicillium species (Figure 1). 1–4 These natural products are synthesized from one molecule each of L-tryptophan, the unnatural amino acid anthranilate (Ant) and an additional natural L-amino acid (proline: 1, 5–8; valine: 2; alanine: 3; and leucine: 4) through the actions of nonribosomal peptide synthetases (NRPSs). For example, psychrophilin B 2 is expected to be cyclized from the L-Trp–L-Val–Ant tripeptidyl thioester attached to the last thiolation domain of the corresponding NRPS. Ant containing fungal peptidyl alkaloids have been well-characterized recently, most of which are structurally complex, multicyclic compounds. 5 Generation of the 13-memberd macrocycle of psychrophilin is however highly unusual, highlighted by formation of the amide bond between the carboxylate of Ant and indole nitrogen of L-Trp, instead of the expected α-NH2 of L-Trp. Interestingly in 1–4, the α-NH2 of L-Trp is oxidized into the rarely observed nitro group; 1–3 whereas in 5–8, the same amino group is acetylated and further methylated. 4 The timing of the L-Trp α-NH2 modification (oxidation or acetylation/methylation) relative to macrocyclization is therefore an interesting biosynthetic question (Figure 2). The α-NH2 group is significantly more reactive in the macrocyclization reaction compared with the indole nitrogen. Nucleophilic cyclization through the α-NH2 (path c) is the canonical NRPS macrocyclization reaction and will lead to formation of a 10-membered macrolactam 11 that may undergo spontaneous quinazoline formation (Figure 2), as observed in other Ant and L-Trp containing tripeptide fungal NRPs such as in fumiquinazoline F. 6, 7 Hence we hypothesize the α-amino group of L-Trp may be oxidized to the nitro group prior to cyclization to prevent formation of 11, and allowing the indole nitrogen to serve as the nucleophile (path b). Alternatively, participation of the indole nitrogen in the cyclization reaction may be entirely controlled by the cyclization domain of the NRPS, and oxidation of the α-NH2 occurs following formation of the cyclic tripeptide 9 (path a). To investigate the biosynthesis of 2 and 3, we sequenced the genome of the producing psychrotoleratnt fungus Penicillium rivulum (IBT 24420) using Illumina HiSeq. 1 The~ 36Mb of genome was assembled and was scanned for NRPS genes using AntiSMASH. 8 A total of 30 NRPS-encoding genes were found, ranging in sizes between mono-module and six-modules. Using the amino acid specificity code determined for adenylation domains that activate Ant, 9 seven distinct modules were found and further analyzed for the potential psy cluster. Two candidate clusters encoded in scaffold 46 and scaffold 182 were found to be the most likely. The cluster encoded in scaffold 182 contains a trimodular NRPS (Pv182-1) and a cytochrome P450 monooxygenase (P450, Pv182-2). While the NRPS and P450 are the minimally required enzymes expected to produce 2 and 3, the Ant-specific A domain was found to reside in the first module of the NRPS, hence inconsistent with the predicted order of amino acid activation (1: L-Trp; 2: L-Val/L-Ala; 3: Ant). Indeed, genetic inactivation of this cluster did not result in the abolishment of psychrophilin production (Supplementary Figure S6). Two separate NRPS-encoding genes are found clustered in scaffold 46 (Figure 2 and Supplementary Table S2), including a dimodule NRPS Pv46-49 with the domain organization of A-PCP-CA-PCP-C (domain abbreviations: A: adenylation; PCP: peptidyl-carrier protein; C: condensation) and a monomodule NRPS Pv46-51 with the domain organization of CA-PCP-CT …