微生物體內二聚化的天然產物到底怎麼咋合成的！

Molecular basis of dimer formation during the biosynthesis of benzofluorene-containing atypical angucyclines

【遇見·按】天然產物領域常見到小分子二聚化的現象，我們在分離純化的時候也能分到很多這樣的產物。那麼二聚化的天然產物在體內到底是如何形成的呢？中科院南海海洋所張長生組剛剛上線並發表在Nat. commun的文章為我們解開了這個謎團...

背景

自然界有很多富含苯並芴結構單元(benzofluorene-containing)的天然產物，如下圖代表性的lomaiviticin A (1), kinamycin A (2), nenestatin A (3), and fluostatins (FSTs, such as4-6)，有很多化合物具有良好的生物學活性。文獻已經報道了化合物

kinamycins (alp), the lomaiviticins (lom), nenestatin A (nes), fluostatins (fls)的生物合成基因簇(其中AlpJ在kinamycin生物合成中負責氧化縮環；Alp1U水解環氧基，Lom6具有相似功能)，但是有些二聚化的天然產物(如1(C2/C2′) 和6(C1/C5′)的C-C鍵)是如何形成的，很少見文獻報道；

[1]Representatives of atypical angucyclines containing a benzofluorene moiety

內容

本文研究人員在Streptomyces albusJ1074中異源表達了fls基因簇(基因簇來自Micromonospora rosariaSCSIO N160)，從40 LStreptomyces albus中分離得到21個產物，部分化合物結構如下[2]；生化分析表徵FlsH為去醯基酶，如下[3]，Docking以及突變顯示FlsH mutants S92A, E115A, and H241F是去醯基化關鍵位點，如圖[4]；自發去醯基化伴隨著二聚化的形成，研究人員還提出了非酶催化的作用機制[5]；最後研究人員利用圖[5]的機制，催化合成了二聚體，如圖[6]。

[2]A partial list of FST-related metabolites isolated from the heterologous hostS. albusJ1074 harboring the fls gene cluster. Selected COSY, HMBC, and NOSEY correlations and the X-ray crystal structure of difluostatin B (15) are also shown. In dimeric compounds, the acceptors and donors are shown in black and blue, respectively.

[3]In vitro characterization of Alp1U, Lom6, and FlsH. a Scheme for Alp1U, FlsH, and Lom6 catalyzed reactions. b HPLC analysis of in vitro reactions of Alp1U, FlsH, and Lom6. Each enzyme assay was performed in 100 μL of 50mM phosphate buffer (pH 7.0) containing 100 μM substrate (4, 5, or 7-10) and 10 μM enzyme (Alp1U, Lom6 or FlsH) for 30 min at 30 °C

[4]Identification of the catalytic triad in FlsH. a A structure model of FlsH highlighting the catalytic triad (Ser-His-Glu) and the docking with FST J (8). b HPLC analysis of enzyme assays with FlsH and three mutated variants. Each enzyme assay was performed in 100 μL of 50mM phosphate buffer (pH 7.0) containing 100 μM 8 and 10 μM enzyme (FlsH or mutated variants S92A, E115A or H241F) for 30 min at 30 °C

[5]Analysis of non-enzymatic reactions of acyl FSTs and the proposed two-step mechanisms.aHPLC analysis of non-enzymatic reactions of acyl FSTs. The general conditions for incubation were set as: substrate (7, 8, 27 or 28) 100 μM, enzyme 10 μM, 30 °C.bProposed mechanisms for the spontaneous deacylation and accompanied dimerization. Chemical structures of compounds27?30are shown in a block as an inset

[6]The proposedp-QM (I)-mediated spontaneous formation of difluostatin B (15) and trifluostatin A (18)

【題外話】本文工作量非常大，需要良好的天然產物化學功底和生物學背景，對工作人員表示敬意和祝賀！

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