Synthetic biocatalyst has been a hot topic for the past 10 years in the world of synthetic chemistry. It has so far been considered a better alternative to metallocatalysis and organocatalysis. The advantage of biosynthesis allows scientists to explore new ways of using enzymes outside of their natural function without polluting solutions with various organic substances.² In what has been described as the third wave of biocatalysts, which started in the mid to late 1990s, scientists have taken to modifying random amino acids in proteins and matching these with variants of the selected enzyme in order to improve enzyme stability and product selectivity. ² This new direction branched out to photobiocatalysis in which semiconductors are used to activate the enzyme.³ However, this approach requires that the co-factor be regenerated, which slows the catalytic process. How can photbiocatalysis and the general third wave of biocatalysts be joined together in order to increase reaction output and specifically produce enantiomer products? In their research paper published with Nature in December 2016, Emmanuel et al. found an answer. In their paper, Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with light, Emmanuel et. al worked with the nicotinamide-dependent enzyme, ketoreductase, and put its co-factor, NAD, through photoexcitation in order to produce a radical intermediate that could be used to in subsequent reactions. The NAD/NADPH co-factors are used as a source of hydrogen atoms and act as photoreductants.
The researchers decided to focus on NAD/NADPH as co-factors because of their photophysical properties. Ketoreducatse (KRED) was used because it relies on this co-factor and is a common enzyme used in biocatalysis to reduce ketones to enantiomerical alcohols via a hydrogen transfer. In it’s ground state, NAD is known as a hydrogen source and a weak single electron reductant. However, when it goes through photo excitation, it becomes a strong single electron reductant, which is used to form the radical intermediate. In this form, it can also reduce multiple functional groups.
Using a variety of KRED variants, the researcher tested a halogenated lactone to see if it will bind well to the enzyme’s active site. With KRED, the halogenated lactone, and NADP in a phosphate buffer, seven enzymes and three variants (KRED-4, KRED-12, and KRED-14) provided the highest yield of the desired product described as lactone 2. This helped the researchers narrow down which enzymes were to be used for the rest of the experiments.
With this information, the researchers hypothesized that NADPH that is not bound to KRED will produce the least amount of lactone 2 compared to a reaction with NADPH was bound to KRED. To test this, the researchers determined the fluorescent lifetime of NADPH with and without a protein. They found that a charge-transfer complex molecule was formed only when NADPH was in the presence of KRED, and that the co-factor is stabilized by the protein. This was determined using ultraviolet/visible-light experiments.
Next, the researchers explored the varying structures of KRED by altering random amino acids in its active site. Combined with site-saturation mutagenesis, the researchers were able to find that when glutamic acid 145 , phenylalanine 147 , and tyrosine 190 were changed to phenylalanine, leucine, and cysteine, respectively, the active site of KRED was enlarged and able to produce an optimal amount of dehalogenated product. This mutation allowed the researchers to manipulate the variants of KRED known to produce high amounts of the desired dehalogenated product.
Next, the researchers confirmed NADPH’s role as a hydrogen source by running the reaction with deuterated isopropyl alcohol in order to form a deuterolactone product with enantioselectivity. With this information available, the researchers were able to work a mechanism in which NADPH goes through photoactivation in which KRED and the substrate bind to form a prochiral radical intermediates that can be used later for subsequent reactions, which can then turn into the desired chiral lactone.
This amazing discovery can help increase production in both chemical laboratories and KRED dependent industrial settings. The researchers also stated that this proposed mechanism can be applied to the beloved co-factor, flavin, which can be used to repair thiamine dimers and the induction of flagellum dependent locomotion.¹
Figure 1. The nicotinamide in 1a is shown going through photoexcitation in order to transform from a weak single-electron reductant to a strong single electron reductant. In its previous form, the nicotinamide served as a hydride source. 1b shows how the enzyme, kedoreductase (KREDs), participates in a reaction between NAD and prochiral ketones in which the hydride from NAD is transferred to pro chiral ketones in order to access chiral alcohols. 1c. shows 1a and 1b together forming a prochiral radical.
1. Emmanuel, M. A., Greenberg, N. R., Oblinksy, D. G. & Hyster, T. K. Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with light. Nature 540, 414–417, 10.1038/nature20569, http://www.nature.com/nature/journal/v540/n7633/full/nature20569.html (2016)
Powered by WPeMatico