Self-sufficient Baeyer-Villiger monooxygenases:: Effective coenzyme regeneration for biooxygenation by fusion engineering
Self-sufficient Baeyer-Villiger monooxygenases:: Effective coenzyme regeneration for biooxygenation by fusion engineering
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DOI：
10.1002/anie.200704630
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Fraaije, Marco W.
中科院分区：
文献类型：
--
作者：
Pazmino, Daniel E. Torres;Snajdrova, Radka;Fraaije, Marco W.
在过去的几年中，工业对进行选择性氧化反应的生物催化剂的兴趣显著增加Baeyer-Villiger单加氧酶（Baeyer-Villiger monooxygenases, BVMOs）是一类用途广泛的酶，可高效催化化学、区域和/或对映选择性氧化反应虽然这些生物催化剂催化的最突出的转化是经典Baeyer-Villiger反应的手性变体[3,4]，但杂原子的氧化和环氧化反应也被报道过这些反应需要化学计量量的O2和NADPH。大规模应用这些反应的一个复杂问题是还原烟酰胺辅酶的高成本为了克服这个问题，已经探索了几种电化学和光化学方法然而，这些方法的效率通常很差。此外，研究表明，BVMOs需要NADP+来维持稳定性和对映选择性催化一种高效且常用的辅酶再生方法是利用整个细胞，特别是结合所需生物催化剂的重组表达该策略已在bvmo介导的野生型菌株[10]的生物转化中实施，并被证明在重组过表达系统中特别成功。[11,12]该方法避免了费力的酶纯化步骤，并利用了宿主的辅酶再生能力。虽然整个细胞已被证明是Baeyer-Villiger氧化的有效催化剂，但它们也有局限性，如细胞毒性、底物/产物对酶的抑制、产物的降解和氧转移速率差近年来，利用分离酶再生辅酶也得到了广泛的研究众所周知的nadph再生酶的例子是乙醇脱氢酶和甲酸脱氢酶亚磷酸酯脱氢酶（PTDH）也被确定为辅酶再生的有效酶有利的热力学平衡常数使亚磷酸酯的氧化成为一个几乎不可逆的过程PTDH对亚磷酸盐的精细选择性也排除了任何副反应，例如当使用醇脱氢酶时可能发生的副反应。这些特性使PTDH成为与BVMOs或其他NAD (P) h依赖性酶结合使用的辅酶再生酶（CRE）的理想候选者。在此，我们报告了一种将氧化还原生物催化剂的催化活性与单个融合蛋白中伴随的辅酶循环结合起来的新方法（方案1）。在过去的十年中，许多融合蛋白标签已经被开发出来。这些标签在生命科学相关的研究和商业活动中被广泛使用。虽然蛋白质融合是一种广泛应用的策略，例如酶纯化（例如使用谷胱甘肽S转移酶（GST）标签）[19]和靶蛋白的亚细胞可视化（例如使用绿色荧光蛋白（GFP）标签）[20]，但这个概念在合成应用的背景下很少遇到。在文献中，只有少数孤立的例子提供证据，证明分离酶的融合可以改善生物催化性能我们在此报道了一些具有代表性的BVMOs的工程，它们与可溶性nadph再生亚磷酸酯脱氢酶共价连接。这种结构使亚磷酸酯作为廉价的牺牲电子供体与整个细胞、细胞提取物和纯化酶一起使用。它……
Over the past few years, industrial interest in biocatalysts that perform selective oxidative reactions has increased significantly.[1] Baeyer–Villiger monooxygenases (BVMOs) have been identified as a highly versatile class of enzymes for the efficient catalysis of chemo-, regio-, and/or enantioselective oxygenation reactions.[2] Although the most prominent transformation catalyzed by these biocatalysts is a chiral variant of the classical Baeyer–Villiger reaction,[3, 4] the oxygenation of heteroatoms and epoxidation reactions have also been reported.[5] Stoichiometric amounts of O2 and NADPH are required for these reactions. A complication for the largescale application of these reactions is the high cost of the reduced nicotinamide coenzyme.[6] To overcome this problem, several electrochemical and photochemical approaches have been explored.[7] However, the efficiency of these approaches is typically poor. Furthermore, it has been shown that BVMOs require NADP+ for stability and enantioselective catalysis.[8] An efficient and commonly used method for coenzyme regeneration employs whole cells, especially in combination with the recombinant expression of the required biocatalysts.[9] This strategy has been implemented in BVMO-mediated biotransformations with wild-type strains [10] and has proved particularly successful with recombinant overexpression systems.[11, 12] The approach avoids laborious enzyme purification steps and exploits the coenzyme regeneration capacity of the host. Although whole cells have been shown to be effective catalysts for Baeyer–Villiger oxidation,[13] they also exhibit limitations, such as cellular toxicity, enzyme inhibition by the substrate/product, degradation of the product, and poor oxygen-transfer rates.[14] Coenzyme regeneration by using isolated enzymes has also been studied extensively in the past few years.[15] Well-known examples of such NADPH-regenerating enzymes are alcohol dehydrogenase and formate dehydrogenase.[16] A phosphite dehydrogenase (PTDH) was also identified as an effective enzyme for coenzyme regeneration.[17] The favorable thermodynamic equilibrium constant makes the oxidation of phosphite a nearly irreversible process.[18] The exquisite selectivity of PTDH for phosphite also precludes any side reactions, such as those that can occur, for example, when an alcohol dehydrogenase is used. These characteristics make PTDH an ideal candidate for use as a coenzyme regenerating enzyme (CRE) in combination with BVMOs or other NAD (P) H-dependent enzymes.Herein, we report a novel approach to the combination of the catalytic activity of a redox biocatalyst with concomitant coenzyme recycling in a single fusion protein (Scheme 1). During the last decade, a number of fusion protein tags have been developed. These tags are used intensely in life-sciencerelated research and commercial activities. Although the fusion of proteins is a widely applied strategy in, for example, enzyme purification (eg the use of glutathione S transferase (GST) tags)[19] and the subcellular visualization of target proteins (eg with a green fluorescent protein (GFP) tag),[20] this concept is hardly ever encountered in the context of synthetic applications. Only a few isolated examples in the literature provide evidence that the fusion of separate enzymes can result in improved biocatalytic properties.[21] We report herein on the engineering of a number of representative BVMOs that are linked covalently to soluble NADPH-regenerating phosphite dehydrogenase. This construct enables the use of phosphite as a cheap and sacrificial electron donor with whole cells, cell extracts, and purified enzyme. It …