酶活性的光调控:用基因编码的光敏剂融合标签使碳糖酶失活

T. Gerlach, Jendrik Schain, Simone Söltl, Morten M. C. H. van Schie, F. Hilgers, N. Bitzenhofer, T. Drepper, D. Rother
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摘要

基因编码的光敏剂能够在光照下产生活性氧,并被广泛应用,特别是在医学领域。在这项工作中,我们设想进一步将这些基因编码的光敏剂应用于多步生物催化中单个酶的光依赖性控制。在级联中应用几种酶的挑战之一是当所有酶同时存在于反应中时,这些生物催化剂对反应中间体的不希望的交叉反应性。作为解决这一问题的一种策略,我们研究了引入基因编码的光敏剂作为融合标签是否可以在成功转化后通过简单地打开光来选择性灭活酶。我们测试了五种不同的光敏剂作为分子生物学融合标签,以灭活巴氏醋杆菌的丙酮酸脱羧酶变体E469G/W543H。二聚光敏剂标签,如枯草芽孢杆菌和恶臭假单胞菌的黄素结合荧光蛋白,显示出与四聚碳连接酶结合形成不溶性蛋白质聚集体的趋势。酶活性在一定程度上保留在这些聚集体中,但处理不溶性聚集体被证明是不可行的。当与四聚体酶融合时,单体光敏剂标签似乎更合适。在黑暗中,与未融合的酶相比,单线态氧光敏蛋白(SOPP3)标记的碳连接酶保留了79%的活性。在蓝光照射下,SOPP3标签显示出最佳的特异性失活,并能够在30分钟内完全失活碳连接酶。因此,SOPP3被视为一种很有前途的光敏剂标签,可应用于未来的多步酶级联,以克服交叉反应性的挑战。
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Photo-Regulation of Enzyme Activity: The Inactivation of a Carboligase with Genetically Encoded Photosensitizer Fusion Tags
Genetically encoded photosensitizers are able to produce reactive oxygen species upon illumination and are exploited in a wide range of applications, especially in the medical field. In this work, we envisioned to further apply these genetically encoded photosensitizers for the light-dependent control of single enzymes in multi-step biocatalysis. One of the challenges in the application of several enzymes in a cascade is the unwanted cross-reactivity of these biocatalysts on reaction intermediates when all enzymes are simultaneously present in the reaction. As one strategy to address this issue, we investigated whether the introduction of genetically encoded photosensitizers as fusion tags would allow the selective inactivation of enzymes after successful transformation by simply turning on light. We tested five different photosensitizers as molecular biological fusion tags to inactivate the pyruvate decarboxylase variant E469G/W543H from Acetobacter pasteurianus. Dimeric photosensitizer tags, like the flavin-binding fluorescent proteins from Bacillus subtilis and Pseudomonas putida showed the tendency to form insoluble protein aggregates in combination with the tetrameric carboligase. Enzyme activity was, to some extent, retained in these aggregates, but the handling of the insoluble aggregates proved to be unfeasible. Monomeric photosensitizer tags appeared to be much more suitable when fused to the tetrameric enzyme. In the dark, the singlet oxygen photosensitizing protein (SOPP3)-tagged carboligase retained 79% of its activity as compared to the unfused enzyme. Upon blue light exposure, the SOPP3 tag showed the best specific inactivation and enabled complete inactivation of the carboligase within 30 min. SOPP3 is thus seen as a promising photosensitizer tag to be applied in future multi-step enzyme cascades to overcome the challenge of cross-reactivity.
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