Abstract
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.
Highlights
Plants use solar energy to synthesize glucose as a chemical energy reserve, in a process called photosynthesis
The artificial photosynthesis system comprises TEOA, eosin Y, NAD+, Rh-based electron mediator (RhM)-azide, and formate dehydrogenase obtained from Thiobacillus sp
As described in the Introduction, we focused on conjugating RhM-azide to TsFDH to facilitate the delivery of NADH regenerated to TsFDH
Summary
Plants use solar energy to synthesize glucose as a chemical energy reserve, in a process called photosynthesis. Efforts have been made to construct artificial photosynthesis systems mimicking natural photosynthesis to synthesize fine chemicals [1,2] They consist of a photocatalytic cofactor regeneration step coupled to a biocatalytic CO2 fixation via a redox enzyme. We hypothesized that conjugation of electron mediator to a permissive site of a redox enzyme will result in a conjugate with retained enzymatic activity and cofactor regeneration capacity. Formate dehydrogense belongs to the superfamily of D-specific 2-hydroxyacid dehydrogenases and catalyzes the oxidation of formate to CO2 It is well known as a standard enzyme for many enzymatic NADH regeneration reactions using formate as a substrate, its unique reverse reaction (CO2 reduction to formate) is beneficial in building a biocatalytic CO2 fixation system due to its specific activity [8]. We tethered TsFDH and (Rh)-coordinated organometallic electron mediator (RhM) to retain significant catalytic activity of TsFDH with the ultimate goal of constructing an efficient artificial photosynthesis system. If not, the regeneration of the Rh would be an alternative option to reduce the cost of cofactor regeneration [17]
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