Identification of 7-Deoxy-desulfo-argino-cylindrospermopsin, the Missing Piece in Cylindrospermopsin Biosynthesis,Journal of the American Chemical Society

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Identification of 7-Deoxy-desulfo-argino-cylindrospermopsin, the Missing Piece in Cylindrospermopsin Biosynthesis
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2022-08-02 , DOI: 10.1021/jacs.2c03932
Annick Méjean ₁ , Olivier Lequin ₂ , Olivier Ploux _{1,

3}

Affiliation

Cylindrospermopsin, a major cyanotoxin, is produced by freshwater cyanobacteria. Its biosynthesis starts from arginine and glycine and involves five polyketide synthases and several tailoring enzymes. We report the identification of 7-deoxy-desulfo-argino-cylindrospermopsin in several cylindrospermopsin-producing cyanobacteria using mass spectrometry experiments. We have purified this new metabolite and established its structure by 1D and 2D NMR spectroscopy using scalar-based ¹H-¹H, ¹H-¹³C, and ¹H-¹⁵N as well as 2D ¹H-¹H ROESY correlation experiments. Using labeled arginines in isotopic incorporation experiments, we have shown that arginine is fully incorporated into 7-deoxy-desulfo-argino-cylindrospermopsin and that the uracil ring of cylindrospermopsin originates from the guanidino moiety of arginine, thus solving a long-standing puzzling question. CyrG and CyrH from the cylindrospermopsin-producing Oscillatoria sp. PCC 6506 were overproduced in Escherichia coli and purified to homogeneity. We showed that CyrG is a zinc-dependent hydrolase, homologous to adenosine deaminases, that transforms 7-deoxy-desulfo-argino-cylindrospermopsin into 7-deoxy-desulfo-cylindrospermopsin and ornithine, with the following kinetic parameters: K_M = 0.21 ± 0.05 μM and k_cat = 0.19 ± 0.02 min^–1. CyrG contained 0.55 mol of zinc per mol of monomer but could be activated by Fe^II or Co^II. CyrH contained almost no metal and showed no such activity even in the presence of excess metal. Using structure-based alignments and secondary structure predictions, we propose that the fifth and last polyketide synthase CyrF in cylindrospermopsin biosynthesis contains an unprecedented C-terminal domain homologous to N-acetyltransferases. We suggest that this domain catalyzes the condensation of the CyrF product with arginine to give 7-deoxy-desulfo-argino-cylindrospermopsin. This would be an unprecedented termination step for a polyketide synthase.

中文翻译：

鉴定 7-脱氧-脱硫-精氨酸-圆柱孢子素，圆柱体孢子素生物合成中的缺失部分

Cylindrospermopsin 是一种主要的蓝藻毒素，由淡水蓝藻产生。它的生物合成从精氨酸和甘氨酸开始，涉及五种聚酮化合物合酶和几种定制酶。我们报告了使用质谱实验在几种产生 cylindrospermopsin 的蓝细菌中鉴定出 7-deoxy-desulfo-argino-cylindrospermopsin。^{我们已经纯化了这种新代谢物，并使用基于标量的1} H- ¹ H、¹ H- ¹³ C 和¹ H- ¹⁵ N 以及 2D ¹ H- ¹通过 1D 和 2D NMR 光谱确定其结构H ROESY 相关实验。在同位素掺入实验中使用标记的精氨酸，我们已经证明精氨酸完全掺入到 7-脱氧-脱硫-精氨酸-圆柱孢菌素中，并且圆柱孢菌素的尿嘧啶环来源于精氨酸的胍基部分，从而解决了一个长期存在的令人费解的问题。CyrG 和 CyrH 来自产生 cylindrospermopsin 的Oscillatoria sp。PCC 6506 在大肠杆菌中过量产生并纯化至同质。我们发现 CyrG 是一种锌依赖性水解酶，与腺苷脱氨酶同源，可将 7-deoxy-desulfo-argino-cylindrospermopsin 转化为 7-deoxy-desulfo-cylindrospermopsin 和鸟氨酸，具有以下动力学参数：K _M = 0.21 ± 0.05 μM 和k _cat = 0.19 ± 0.02 分钟^–1。CyrG 每摩尔单体含有 0.55 摩尔锌，但可以被 Fe ^II或 Co ^II活化。CyrH 几乎不含金属，即使在过量金属存在的情况下也没有显示出这种活性。使用基于结构的比对和二级结构预测，我们提出圆柱体孢子素生物合成中的第五个和最后一个聚酮化合物合酶 CyrF 包含一个前所未有的与N-乙酰转移酶同源的 C 末端结构域。我们建议该结构域催化 CyrF 产物与精氨酸的缩合以产生 7-脱氧-脱硫-精氨酸-圆柱体视蛋白。这将是聚酮化合物合酶前所未有的终止步骤。

更新日期：2022-08-02

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