Lim Sung In, Yoon Sungho, Kim Yong Hwan, Kwon Inchan
Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, USA.
Department of Bio & Nano Chemistry, Kookmin University, 861-1 Jeoungnung-dong, Seongbuk-gu, Seoul 136-702, Korea.
Molecules. 2015 Apr 7;20(4):5975-86. doi: 10.3390/molecules20045975.
Photosynthesis consists of a series of reactions catalyzed by redox enzymes to synthesize carbohydrates using solar energy. In order to take the advantage of solar energy, many researchers have investigated artificial photosynthesis systems mimicking the natural photosynthetic enzymatic redox reactions. These redox reactions usually require cofactors, which due to their high cost become a key issue when constructing an artificial photosynthesis system. Combining a photosensitizer and an Rh-based electron mediator (RhM) has been shown to photocatalytically regenerate cofactors. However, maintaining the high concentration of cofactors available for efficient enzymatic reactions requires a high concentration of the expensive RhM; making this process cost prohibitive. We hypothesized that conjugation of an electron mediator to a redox enzyme will reduce the amount of electron mediators necessary for efficient enzymatic reactions. This is due to photocatalytically regenerated NAD(P)H being readily available to a redox enzyme, when the local NAD(P)H concentration near the enzyme becomes higher. However, conventional random conjugation of RhM to a redox enzyme will likely lead to a substantial loss of cofactor regenerating capacity and enzymatic activity. In order to avoid this issue, we investigated whether bioconjugation of RhM to a permissive site of a redox enzyme retains cofactor regenerating capacity and enzymatic activity. As a model system, a RhM was conjugated to a redox enzyme, formate dehydrogenase obtained from Thiobacillus sp. KNK65MA (TsFDH). A RhM-containing azide group was site-specifically conjugated to p-azidophenylalanine introduced to a permissive site of TsFDH via a bioorthogonal strain-promoted azide-alkyne cycloaddition and an appropriate linker. The TsFDH-RhM conjugate exhibited retained cofactor regenerating capacity and enzymatic activity.
光合作用由一系列由氧化还原酶催化的反应组成，利用太阳能合成碳水化合物。为了利用太阳能，许多研究人员研究了模拟自然光合酶促氧化还原反应的人工光合作用系统。这些氧化还原反应通常需要辅因子，由于其成本高昂，在构建人工光合作用系统时成为一个关键问题。已证明将光敏剂和基于铑的电子介体（RhM）结合可光催化再生辅因子。然而，要维持用于高效酶促反应的高浓度辅因子，需要高浓度的昂贵RhM，这使得该过程成本过高。我们假设将电子介体与氧化还原酶结合会减少高效酶促反应所需的电子介体数量。这是因为当酶附近的局部NAD（P）H浓度升高时，光催化再生的NAD（P）H很容易被氧化还原酶利用。然而，传统的将RhM随机结合到氧化还原酶上可能会导致辅因子再生能力和酶活性的大量损失。为了避免这个问题，我们研究了将RhM生物结合到氧化还原酶的允许位点是否能保留辅因子再生能力和酶活性。作为一个模型系统，将RhM与一种氧化还原酶——从硫杆菌属KNK65MA（TsFDH）获得的甲酸脱氢酶结合。通过生物正交应变促进的叠氮化物-炔烃环加成和合适的连接子，将含叠氮基团的RhM位点特异性地结合到引入TsFDH允许位点的对叠氮苯丙氨酸上。TsFDH-RhM共轭物表现出保留的辅因子再生能力和酶活性。
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