新颖的韧革菌素环合酶及其分子机制研究

Vibralactone, a rare 4/5 fused bicyclic β-lactone, was isolated from the basidiomycete fungus Boreostereum vibrans and found to inhibit pancreatic lipases highly relevant to fat absorption. Inspired by its unique chemical structure and clinical potential for the treatment of obesity, we have recently established the biosynthetic origin of vibralactone and obtained evidence for its biosynthetic pathway that includes several very interesting reactions that may involve unusual enzymes. Our results unequivocally demonstrate that the bicyclic lactone core of vibralactone is derived neither from a seemingly plausible polyketide nor a sesquiterpenoid origin, but instead a shikimate pathway, and could be attributed to a cyclase-catalyzed intramolecular cyclization of the seven-membered ring intermediate. The establishment of its biosynthetic pathway will be valuable for future elucidation of the molecular mechanism for vibralactone biosynthesis. Furthermore, very little has been done on the biosynthetic machineries for bioactive natural products isolated from basidiomycete fungi (also known as mushrooms) in general. Here we aim to track and purify the vibralactone cyclase through an activity-guided purification and protein profiling. Based on heterologous expression and functional characterization of putative enzyme genes, we can identify the enzyme specific for intramolecular cyclization to vibralactone. We hope to provide a glimpse at how the vibralactone cyclase catalyzes the unusual intramolecular C-C cyclization by enzyme kinetics and point mutation experiments. This study not only enables some insight into the understanding of natural product biosynthesis in higher fungi but also supports the wisdom of applying combinational chemistry / synthetic biology for the generation of novel vibralactone analogues, thereby facilitating drug development efforts based on these privileged natural product scaffolds as bioactive labels and lead compounds.

韧革菌素(Vibralactone)是高等真菌褐盖韧革菌(Boreostereum vibrans)产生的活性小分子，其4/5融合双环内酯骨架结构在天然产物中十分罕见。韧革菌素不仅结构新颖，而且显著抑制与脂肪吸收相关的胰脂肪酶活性。最近我们证实了韧革菌素不同寻常的生物合成途径及新颖的环合酶反应。本研究运用酶活性导向分离纯化与蛋白组学技术，分离鉴定具有韧革菌素环合酶活性的蛋白组分；根据基因组、转录组与代谢谱分析，挖掘可能的目标基因；通过异源表达以及酶功能的体外与体内鉴定，进行韧革菌素环合酶基因的功能鉴定；通过酶动力学与氨基酸定点突变研究，解析韧革菌素环合酶新颖的催化机理。研究韧革菌素环合酶新颖的分子机制与催化机理，将为运用组合化学/合成生物学等前沿技术设计和创造新的生物合成途径与新的活性分子奠定基础，进一步拓展韧革菌素类化合物作为分子探针及先导分子在药物发现和化学生物学领域的应用前景。

具有融合双环内酯骨架结构的韧革菌素 (Vibralactone) 不仅化学结构新颖独特，而且显著抑制胰脂肪酶活性。前期我们证实了韧革菌素不同寻常的生物合成途径及新颖的环合酶反应，本研究通过前体类似物喂养与转化产物分离鉴定、酶活性导向的蛋白质分离纯化与蛋白组学技术、基因挖掘与酶活检测等，发现并鉴定了高等真菌褐盖韧革菌中催化七元内酯环发生分子内环合生成韧革菌素的环合酶为α/β-水解酶家族成员；通过酶学特性、氨基酸定点突变、晶体结构等研究，揭示了韧革菌素环合酶 (Vibralactone cyclase, 简称VibC) 新颖的催化机理，即利用丝氨酸-组氨酸-天冬氨酸三联体催化底物氧杂环庚三烯-2(3H)-酮产生亲核烯醇阴离子/碳负离子，从而引发分子内aldol反应或电环合反应形成韧革菌素的双环内酯骨架。韧革菌素环合酶的结构和催化机理完全不同于经典的aldol酶，也有别于聚酮类aldol环合酶；尽管其催化位点与水解酶相似，但两者的催化机制各不相同。韧革菌素环合酶是首个自然酶促aldol反应或电环合反应的水解酶，它的发现丰富了天然产物生物合成中aldol反应酶及周环酶的分子机制与催化机理，扩展了水解酶的自然催化反应类型，为合成生物学设计和创造新的生物合成途径与新的活性分子奠定了基础。