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Enzyme beats catalysts at oxidation game

Directed evolution yields enzyme that oxidizes alkenes in a unique way

by Stu Borman
October 12, 2017 | A version of this story appeared in Volume 95, Issue 41

Directed evolution substitutions are distributed all around the structure of the enzyme that was modified.
Credit: Science
Changing 12 amino acids (red circles) in P450LA1 yielded the enzyme aMOx. Heme group is black, blue, and red stick structure.

Oxidizing the end carbon atom of a terminal alkene to form an aldehyde, a process called anti-Markovnikov alkene oxidation, is difficult because the reaction is energetically unfavorable. The preferred process, in terms of energetics, is Markovnikov selectivity—oxidation of the substrate’s next-to-last carbon, forming a ketone instead of an aldehyde.

Chemists have long wanted to find better ways to achieve anti-Markovnikov selectivity in alkene oxidations—for example, to convert the aldehyde products to linear alcohols, which are commonly used in flavorings, perfumes, lubricants, and cosmetics. Catalysts exist that can perform the tricky anti-Markovnikov oxidations, but they are very unproductive and are not enantioselective.

Frances H. Arnold and coworkers at Caltech thought a modified enzyme might succeed where traditional catalysts have struggled. The team now reports using directed evolution, an iterative process of protein mutation and screening for activity, to identify amino acid substitutions that convert an enzyme into a predominantly anti-Markovnikov alkene oxidation catalyst (Science 2017, DOI: 10.1126/science.aao1482).

The evolved enzyme, called aMOx, is fairly productive, catalyzing about 3,800 oxidation reactions, or turnovers, before running out of steam, compared to fewer than 10 for earlier anti-Markovnikov oxidation catalysts. In conjunction with other reagents and catalysts, the enzyme could one day convert terminal-alkene substrates to a variety of aldehyde, alcohol, and amine products for commodity and fine chemical, pharmaceutical, and agrochemical use.

Reaction scheme shows difference between Markovikov (ketone formation) and anti-Markovnikov (aldehyde formation) of terminal alkenes.
Markovnikov oxidation of terminal alkenes (left) to form ketones (center) is energetically favored, whereas anti-Markovnikov reactions leading to aldehydes are more difficult.

The Caltech researchers started with an iron-based cytochrome P450 enzyme called P450LA1because it catalyzes a related reaction, metal-oxo alkene oxidation, in which a metal-linked oxygen attacks the double bond, typically forming an epoxide. An earlier study by another group had found that P450LA1 also made 19% aldehyde side-product by opening the epoxide. In the new study, postdoc Stephan C. Hammer, now at the University of Stuttgart, found that the enzyme actually made 45% aldehyde directly, without going through an epoxide intermediate.

The researchers then used 10 rounds of directed evolution, with styrene as a substrate, to boost anti-Markovnikov selectivity from 45% to 81%, in aMOx. aMOx works well because it stabilizes high-energy radical and carbocation intermediates created when its iron-oxo group attacks styrene’s terminal alkene, allowing anti-Markovnikov aldehyde formation to predominate.

It took 12 mutations distributed throughout the protein to transform P450LA1 into aMOx. “No one could explain how these substitutions confer this reaction selectivity, much less predict them,” Arnold says. “This illustrates the power of directed evolution to create catalysts that have eluded chemists.”

Key advantages of the evolved enzyme include its enantioselectivity and its turnover number, “which probably still isn’t sufficient for commercial viability but pretty respectable,” says John T. Groves of Princeton University, who specializes in biomimetic catalysis.

There’s currently a push in academia and industry to replace conventional catalytic reactions with enzymatic processes that use aqueous solvents at ambient temperatures and pressures, work readily in flow systems, and don’t require catalyst separations, Groves says. “These days, a tie will go to the biocatalyst for those reasons. The new study is an important step in the right direction.”

This article has been translated into Spanish by and can be found here.


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