A Light at the End of the Tunnel – Photoactive Co-factors

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)

2. Bornscheuer, U. T. et al. Engineering the third wave of biocatalysis. Nature 485, 185–194, 10.1038/nature11117, http://www.nature.com/nature/journal/v485/n7397/full/nature11117.html (2012)

3. Maciá-Agulló, J. A., Corma, A. & Garcia, H. Photobiocatalysis: The power of combining Photocatalysis and enzymes. Chemistry – A European Journal 21, 10940–10959, 10.1002/chem.201406437 (2015)

12 Replies to “A Light at the End of the Tunnel – Photoactive Co-factors

  1. Hi Aisha. I found the mutational studies on KRED interesting. How did the glutamic acid 145 , phenylalanine 147 , and tyrosine 190 mutations selectively facilitate the production of the dehalogenated product? In other words, how is the reductive enzyme causing a dehalogenation? Furthermore, I am also curious about the relationship between the mutational and photosensitation studies. Are the mutational studies independent of the photosensitation studies or is the photo-excited NAD co-factor still necessary for the desired reaction?

    1. Hi Melanie,
      The researchers found that mutation of tyrosine 190 allowed LKADH, the enzyme used throughout the experiments, to reduce sterically demanding substrates like bulky ketones and allowed dehalogenation activity, but with low product yield. When they also mutated glutamic acid 145 and phenylalanine 147 to phenylalanine and leucine, respectively, they found that this faciliated the production of the dehalogenated product at a yield of 72%. The three mutations also resulted in a larger active site, which allowed dehalogenation activity.
      The photo-excited NAD is still necessary for the desired reaction because under a photoexcited state, ketoreductase (NAD dependent) can be transformed to be the initiator for a radical species as well as a chiral source for hydrogen atoms, which is what the researchers were looking for.

  2. This study has extremely high value due to the fact some racemic mixes are completely harmless, while other mixes have one harmless enantiomer and the other can cause serious problems. Thalidomide was a drug taken by pregnant women and the one enantiomer caused severe birth defects. Methods of controlling enantiomer products are very crucial. As for the experiment on hand, the discovery of those mutations in the active site to amplify productivity was quite a find. I question if there were any mechanistic details that helped explain why these mutations are better. Another small question I have is the use of the photoexcitation. It wasn’t crystal clear to me if it was used to be able to detect the chirality of the molecule or if it also added energy to push the reaction.

    1. Hi Nick,
      The researchers found that a the mutations allowed the for a larger active site, which in turn allowed dehalogenation activity. Photoexcitation helps overcome the slow regeneration of a co-factor by forming prochiral radical intermediates, which can be used later for other reactions and can be turned into the desired product, a chiral lactone.

  3. I really enjoyed reading this article. As soon as I have finished reading, I instantly thought of how the enzymes, after light energy is absorbed, help the chloroplasts membrane establish electron flow to synthesis ATP. Most of these reactions are so similar to what happens inside the mitochondria to synthesis ATP. Also, Ketoreductase enzymes (KRED) could really help us understand other reactions since they can be mutated to catalyse reactions other than the natural ones. Just two question for you how did they determine if the affinity or the rate of the hydrogen transfer was affected by the mutations that they generated (glutamic acid 145, phenylalanine 147, and tyrosine 190 were changed to phenylalanine, leucine, and cysteine respectively)? Also, do you think the created mutations help stabilize the cofactor any better than the wild type? Good work!

    1. Hi Renan,
      You made some great connections! When running the mechanistic experiments, the researchers used deuterated isopropyl alcohol which in turn generated deuterated NADPH in situ. The result was that the primary product was deuterolactone with a recorded 92% deuterium incorporation. This confirmed that the mutations they made affected hydrogen output.
      From the results, it does seem that the mutations stabilized the cofactor better than the wild type considering they were trying to create a greater output than what the wild type enzyme could handle. The mutations created a larger active site, which supported dehalogenation activity.

  4. After reading the article, I was interested in how the enzyme’s active site would tolerate the prochiral radical that is formed in the reaction. I understand that the authors believe that the process is so quick that the prochiral radical does not survive for long, but I still am curious if prochiral radical can bind to the enzyme, or do other undesirable activities, within that short period of time, as radicals are such unstable molecules. Is it possible that author’s engineering of a larger active site might mitigate any ill effects of the prochiral radical, as it might have more space to move about without pumping into residues and reacting with them? Even if this is not a problem on the scale that the authors are studying the reaction under, do you think that on an industrial scale the prochiral radical might become more of an issue for the enzyme?

    1. Hi Brock,
      I actually had the same concerns when reading this paper. I think it is possible that there could be ill side effects of the radical if it survives longer than intended. However, it seems that there is a small chance of this happening based on the data presented in the paper. If put into industrial scale, I’d assume that there would be a chance of side reactions with the prochiral radical. I did not find any resource or follow up discussing these concerns. I hope it is something they would look at in the future.

  5. Hi Aisha!

    You mention that this mechanism can be applied to flavin, which you say is involved in reparation of thymine dimers. Generally, while reading your post, my thoughts revolved around the possible dangers of radical intermediates and causing oxidative stress for cells. I mentioned the thymine dimers particularly because it seems that reactive radical intermediates can be dangerous for DNA structure. Is it reasonable to wonder if using photoexcitation to make nicotinamide a better single electron reductant can be harmful?

  6. Hi Aisha! I think this spotlight was incredibly fascinating for many reasons. As we learned in experimental biochemistry, the use of photo reactive compounds is important for determining both mechanism and structure of an enzymatic reaction. Using these photoreactive cofactors seems to be incredibly important in determining the absolute configuration of a product and if the reaction will need to be manipulated in order to produce one enatiomer over the other. History shows us why it is so incredibly important to use one enantiomer over the other in drug therapies.

  7. Hi Aisha,
    Thanks for sharing this article! I think it’s really great to see a more chemical side of biochemistry. Keeping in line with that, I would be interested to see if physical chemistry could illuminate more as to a pattern of excitation when photoexciting and maybe even be used to expand on this knowledge using different energies of light to accomplish different outcomes. I think it would be interesting to see if using a more mathematical approach may help with screening different mutations and reactions rather than the guess and check method.

  8. We often talk a lot about how pathways in biochemistry often differs greatly from organic chemistry because the organic pathways would kill an organism. I thought it was interesting that the authors used NADPH/NAD for its photophysical properties when we discuss in class its important in many metabolic pathways. In addition, the fact that there was stereoselectivity further shows the importance of discovering new ways to investigate biosynthetic pathways.

Leave a Reply

Your email address will not be published. Required fields are marked *