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Scientists push enzyme evolution into high gear

Scientists at Brookhaven National Laboratory have found a way to make a plant enzyme that is 100 times more efficient than similar enzymes found in nature

A model of the engineered enzyme's active site. The green lines represent the backbone of the amino acid chain. The yellow lines show amino acid side chains from the original enzyme; the purple lines are the evolved side chains. The white ghostly molecule is the fatty acid. The red spheres are iron atoms. (Image by Bill McGrath)

The Brookhaven study offers insight into how enzymes evolve and may one day lead to methods to boost production of other useful plant products.

"Plants make many valuable compounds, but often in small quantities," says John Shanklin, the lead biologist on the study. Examples could include medicinal compounds and oils that may be useful as raw materials for industrial processes.

Shanklin suggests that the reason for such poor production in nature is that the enzymes responsible are newly evolved. "That may seem strange, because many people associate evolution with improvement. But when enzymes evolve new functions, they almost always lose efficiency," he says.

Enzymes are proteins that speed up chemical reactions by bringing the reacting molecules together like pieces of a puzzle. Like all proteins, they're made of chains of building blocks called amino acids, folded in a precise way to give the enzyme its three-dimensional shape.

In nature, new enzymes arise from random mutations in the genes that code for the amino-acid sequence. Most changes have no effect. A very small percentage improve the enzyme or give it a new function. But more often the changes deform the enzyme, making it ineffective or unstable, Shanklin says. Over hundreds or even millions of years, natural selection might improve the new enzyme. But Shanklin and his team thought there might be a more direct way—to evolve a better enzyme in the laboratory.

The method

Shanklin and fellow Brookhaven biologist Ed Whittle were interested in making a more efficient fat-modifying enzyme with properties similar to enzymes they had isolated from milkweed plants and cat's claw vines. These slow-acting enzymes had evolved from a similar enzyme that was much more efficient, but modified a larger fat. To figure out how to turn the parent enzyme into one that could modify smaller fats without losing efficiency, the first step was to find out which amino acids in the parental enzyme could change the enzyme's specificity.

The scientists used a technique to introduce mutations, one or two at a time, into the ancestral enzyme gene. They then screened the resulting enzymes to identify those that could modify smaller fats. By sequencing the genes from those varieties, they found six amino acid locations (out of 350 total) capable of changing the enzyme specificity.

Shanklin and his team synthesized genes for all the possible combinations of nature's 20 amino acids in those six spots, for a total of 64 million varieties, and then inserted these genes into a bacterial strain that required the new enzyme for survival. Most of the bacterial cells died because they lacked changes that were necessary to make the crucial enzyme. Those that did produce the enzyme thrived, and their genes revealed the identity of key mutations.

Now that the scientists knew which mutations would lead to the new enzyme, they were able to make only those changes in the original gene. The resulting enzyme turned out to be 100 times more efficient than the two varieties they had isolated from nature.

"We've now put this gene into Arabidopsis plants-the experimental fruit flies of plant science--and it works very well," Shanklin says.

This process of "tuning up" enzymes, Shanklin says, might be useful to produce large quantities of other plant products. For example, he says, one day, plant products might be used to meet our growing need for industrial raw materials--perhaps even taking the place of petroleum-based chemicals.

"The idea is to grow natural resources instead of taking them from a nonrenewable source," he says.—Karen McNulty Walsh



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