Ordering the reductive and cytochrome P450 oxidative steps in demethylsterigmatocystin formation yields general insights into the biosynthesis of aflatoxin and related fungal metabolites
Ordering the reductive and cytochrome P450 oxidative steps in demethylsterigmatocystin formation yields general insights into the biosynthesis of aflatoxin and related fungal metabolites
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DOI：
10.1021/ja0455188
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发表时间：
2005-03-23
影响因子：
15
通讯作者：
Townsend, CA
中科院分区：
文献类型：
--
作者：
Henry, KM;Townsend, CA
有效的环境致癌物质黄曲霉毒素B1的生物合成涉及比第一聚酮中间体约15个步骤。其中最重要的是将杂色蒽醌A重排成去甲基杂色藻毒素。遗传证据有力地表明，这一过程需要两种酶，一种是细胞色素P450，即AFIN，另一种可能是NADPH依赖的氧化还原酶，AFIM。考虑到在这种骨架重排中明显的整体氧化还原变化，必然会发生两轮氧化和还原。早期的实验表明，杂色苷A的还原脱氧不是第一步。在本报告中，我们考虑了一种机制替代方案，即AFIM介导的还原反应是这三个反应中的最后一个反应，而不是形成xanthone中间体。为此，采用全合成的方法合成了9-羟基二氢脱甲基杂色曲霉毒素及其类似物--黄曲霉毒素前体9-脱氧。在最终分离“角形”合成的xanthone靶标的过程中，发现酸催化促进了它们的异构化为热力学上有利的“线性”xanthone。用途径第一步阻断的寄生曲霉突变株进行9-羟基和9-脱氧氧杂多酮的全细胞和地面细胞培养，并检测它们支持黄曲霉毒素产生的能力。9-脱氧杂环己酮显著提高了真菌毒素的水平。另一方面，在黄曲霉毒素的生物合成过程中，9-羟基黄原酮的黄曲霉毒素含量没有明显增加，这表明在黄曲霉毒素的生物合成过程中，黄原酮前体在C-9处没有发生还原脱氧反应。以前的研究和本文的实验施加的限制有助于消除杂色素A简单和直观地转化为去甲基杂色藻毒素，并不可避免地导致更微妙的氧化-还原-氧化反应序列。以前令人费解的观察到杂色甲素重排为去甲基杂色藻毒素过程中广泛的A环氢交换，现在用一种与现有数据一致的新机制解释了这一现象。我们认为，P450介导的芳基环氧化(AFIN)首先破坏花色苷A的芳香族A环，氧杂环的开放使A环质子交换，以及随后AFIM介导的还原步骤。第二个周期的P450氧化(AFIN)，这一次是Baeyer-Villiger裂解，使脱羧基和形成去甲基杂色曲霉毒素。描述了作为这一提议基础的机械论和立体电子学原理，并且可能被证明是一般的，如在其他四种真菌的蒽醌双右箭头xanthone转化的生物遗传假说中所示。
The biosynthesis of the potent environmental carcinogen aflatoxin B1 involves ca. 15 steps beyond the first polyketide intermediate. Central among these is the rearrangement of the anthraqinone versicolorin A to the xanthone demethylsterigmatocystin. Genetic evidence strongly suggests that two enzymes are required for this process, a cytochrome P450, AfIN, and a probable NADPH-dependent oxidoreductase, AfIM. Given the overall redox change evident in this skeletal rearrangement, two rounds of oxidation and a reduction necessarily occur. Earlier experiments indicated that reductive deoxygenation of versicolorin A is not the first step. In the present report we consider a mechanistic alternative that AfIM-mediated reduction is instead the last of these three reactions prior to formation of the xanthone intermediate. To this end, 9-hydroxydihydrodemethylsterigmatocystin was prepared by total synthesis as was its 9-deoxy analogue, an established aflatoxin precursor. During the final isolation of the "angular" synthetic xanthone targets it was found that acid catalysis promoted their isomerization to thermodynamically favored "linear" xanthones. Whole-cell and ground-cell incubations of the 9-hydroxy- and 9-deoxyxanthones were conducted with a mutant strain of Aspergillus parasiticus blocked at the first step of the pathway and examined for their ability to support aflatoxin production. The 9-deoxyxanthone gave dramatically enhanced levels of the mycotoxin. The 9-hydroxyxanthone, on the other hand, afforded no detectable increase in aflatoxins above controls, indicating that reductive deoxygenation at C-9 of a xanthone precursor does not take place in aflatoxin biosynthesis. Constraints imposed by earlier studies and the experiments in this paper serve to eliminate simple and intuitive conversions of versicolorin A to demethylsterigmatocystin and lead inescapably to a more subtle reaction sequence of oxidation-reduction-oxidation. Previous puzzling observations of extensive A-ring hydrogen exchange in the course of the rearrangement of versicolorin A to demethylsterigmatocystin have now been explained by a new mechanism that is consistent with all extant data. We propose that P450-mediated aryl epoxidation (AfIN) initially disrupts the aromatic A-ring of versicolorin A. Oxirane opening enables A-ring proton exchange, as does the subsequent AfIM-mediated reductive step. A second cycle of P450 oxidation (AfIN), this time a Baeyer-Villiger cleavage, enables decarboxylation and the formation of demethylsterigmatocystin. Mechanistic and stereoelectronic principles that underlie this proposal are described and may prove general as illustrated in biogenetic hypotheses for four other fungal anthraquinone double right arrow xanthone transformations.