UPTON, NY -- Scientists at the U.S. Department of Energy's Brookhaven National Laboratory have found a way to make a plant enzyme that is 100 times more efficient than similar enzymes found in nature. The research, described in the June 15, 2001 issue of The Journal of Biological Chemistry, 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. Though not the direct focus of Shanklin's work, 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. "Could we evolve a better enzyme in the laboratory?" he asked.
Shanklin and fellow Brookhaven biologist Ed Whittle were interested in making a more efficient fat-modifying enzyme with properties similar to fat-modifying 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.
They then synthesized genes for all the possible combinations of nature's 20 amino acids in those six spots, for a total of 64 million varieties. Then they 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 the scientists 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. Now that's evolutionary thinking!
This work was funded by the U.S. Department of Energy, which supports basic research in a variety of scientific fields.
The U.S. Department of Energy's Brookhaven National Laboratory conducts research in the physical, biomedical, and environmental sciences, as well as in energy technologies. Brookhaven also builds and operates major facilities available to university, industrial, and government scientists. The Laboratory is managed by Brookhaven Science Associates, a corporation founded by Stony Brook University and Battelle, a nonprofit applied science and technology organization.
Materials provided by Brookhaven National Laboratory. Note: Content may be edited for style and length.
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