Researchers develop a method that gives enzymes the ability to catalyze reactions new to nature

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Enzymes are the catalytic workhorses of biology, linking molecules together, breaking them apart, and remodeling them into processes vital to everything from digestion to respiration. Its availability, efficiency, and specificities have long made it popular for interactions outside biological systems as well, including those involved in food and detergent preservation and disease diagnosis.

“Enzymes are nature’s signature catalysts,” said Yang Yang, associate professor of chemistry at the University of California, Santa Barbara. “They can catalyze reactions with amazing selectivity.” Efforts over the past three decades have also led to the development of customized enzymes – enzymes that have rapidly evolved for purpose-directed, targeted interactions with specific molecules, resulting in high yields of desirable products with unparalleled selectivity.

However, Yang added, the reactions that enzymes can allow are relatively limited — a fairly small ammunition for their powerful ability to efficiently manufacture products at lower material, energy and environmental costs.

To bridge this gap and combine the best of both worlds – diversity and selectivity – Yang and his research team developed a method in which specific enzymes can be coaxed into facilitating beneficial interactions not previously observed in the biological world, thus expanding their repertoire and opening up possibilities for never-before-seen processes by enzymes.

said Yang, who along with colleagues at the University of California, Santa Barbara and the University of Pittsburgh, authored a research paper that appeared in the journal. Science.

3D chemistry

Stereotactic chemistry (also known as 3D chemistry) is necessary to control the bioactivity of small-molecule drugs. Most biomolecules, including DNA and proteins, are viral, which means that they are asymmetric in structure.

“It’s like your left hand and your right hand: they look alike but aren’t superimposable, which means they’re flexible,” Yang explained. “To effectively interact with these chiral biomolecules, small-molecule drugs must be designed using specific stereochemistry. Often, one analogue of a chiral drug molecule is highly effective, while the other analogue is ineffective or even toxic.”

The most efficient way to create such valuable chiral molecules, he said, relies on asymmetric catalysis, a process in which a dedicated catalyst selectively produces one symmetric (non-combinable molecule) instead of the other. Unfortunately, many challenges remain in the field of asymmetric catalysis. In particular, a class of widely used reactions—that is, radical reactions or reactions involving open-shell intermediates—has not yet undergone asymmetric catalysis. This problem has long eluded synthetic chemists.

“Organic radicals are very common, and they react very actively in synthetic chemistry,” Yang said. “However, it is known that controlling the stereochemistry of these reactions is very difficult.”

He explained that two problems arise. The first is that the radical, once formed, does not usually react tightly with the catalyst.

“Therefore, there is no way to impose holographic control over these radical formations that are mediated by links,” he said.

Second, there is often an activity-selective trade-off.

“If you have a very active species, it will be relatively difficult for you to control the selectivity of reactions involving these intermediates. So there is usually a trade-off,” Yang said.

the solution? Directed evolution – development of an enzyme to be able to restrain a radical.

Inspired by 2018 Nobel Prize-winning chemical engineer Francis Arnold, who was Yang’s postdoctoral advisor, the team conducted iterative rounds of evolution and examined the P450 cytochromes. The superfamily of metalloenzymes is found in all kingdoms of life that contain heme – an iron-containing molecule – which is necessary for catalysis.

“Guided evolution uses these rounds of mutation and screening to improve enzyme functions,” Yang explained. “In the process, we are creating a huge library of enzyme variants by manipulating DNA.” With a DNA library of target interactions, researchers can express and screen for protein mutations to help identify promising enzyme promoters. The improved enzyme then becomes the parent in the subsequent engineering round. In this way, through iterative cycles of mutation and screening, optimal enzyme activity and selectivity are reached.

Using this method, the researchers were able to repurpose an enzyme to perform an “abnormal biocatalytic reaction, i.e., steric selective atom transfer radical hydrolysis”, by combining the power of artificial and nature-controlled catalysis with enzymatic catalysis.

This new ability opens up many possibilities, including a variety of molecules that newly developed enzymes can affect.

“The overall goal is to apply the biocatalysts we develop to the pharmaceutical and agrochemical industries,” Yang said. “Ultimately, using the new tools, we will be able to develop valuable drugs and herbicides that will be beneficial to our society.”

Researchers access each of the viewers by varying the reaction time

more information:
Qi Zhou et al, The radical cycle of spaced-atom transfer by engineered P450 cytochromes, Science (2021). DOI: 10.1126 / science.abk1603

Offered by University of California – Santa Barbara

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