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Study Finds Plant Enzyme Function Changes with Location in Cell

First example of location-dependent production of alternate products by single enzymes

Photo of John Shanklin and Ingo Heilmann

Click on the image to download a high-resolution version. John Shanklin (top) and Ingo Heilmann. (Click image to download hi-res version.)

UPTON, NY - Scientists have long thought that individual enzymes have specific, single jobs dependent on their molecular shape. According to this premise, enzymes could only evolve to perform new functions by accumulating several shape-changing mutations, which can take thousands of generations. Now, scientists at the U.S. Department of Energy's Brookhaven National Laboratory have discovered another factor that can change several plant enzymes' functions instantaneously: their location within the cell. Depending on where these enzymes end up, they produce slightly different products.

"It looks like the old axiom of what's important in real estate - location, location, location - holds for some enzymes as well," says Brookhaven biochemist John Shanklin, author of a paper describing the first example of such location-dependent enzyme function in the July 13, 2004, issue of the Proceedings of the National Academy of Sciences.

Multifunctional enzymes such as these could substantially expand the diversity of metabolic products available to a cell, thereby increasing the organism's ability to adapt to changing conditions, Shanklin says. They may also offer scientists new ways to tailor plant products to meet specific needs, such as growing crop plants that make different, perhaps healthier oils.

Shanklin and his research associate Ingo Heilmann were studying a particular class of enzymes known as desaturases in Arabidopsis, the equivalent of the fruit fly for plant scientists. Desaturases remove hydrogen atoms from fatty acid chains to create carbon-carbon double bonds, desaturating the fat. While using yeast cells to determine the functions of several newly discovered Arabidopsis desaturases, Heilmann and Shanklin noticed that one of the enzymes appeared to operate differently in yeast than it did in the plants: It inserted a double bond in a different place along the fatty acid carbon chain.

The scientists knew that, in the plant cells, the enzyme is tagged with an "address signal" that directs it to the plant cells' chloroplasts, the green organelles within plant cells where photosynthesis takes place. When Heilmann removed this address signal, the plant enzyme no longer went into the chloroplast, but instead entered the endoplasmic reticulum, a membranous structure within the cell. In that location, the enzyme desaturated fatty acids the same way it did in yeast. Conversely, when the scientists added the chloroplast address signal to several desaturases that normally operate in the endoplasmic reticulum, they instead traveled into the chloroplasts and operated like the desaturases normally found there.

"So instead of waiting for multiple mutations to accumulate to produce a new function, one simple addition or deletion of an address signal - a single mutation - could change the enzyme's product instantaneously," Shanklin says. But the scientists still didn't know how the change in location produced the change in enzyme function.

They suspected that lipid head groups - a sort of "scaffold" that links the fatty acids and takes different forms in the chloroplasts and endoplasmic reticulum - might play a role. By adding the gene responsible for building the chloroplast lipid head group to yeast cells, the scientists were able to produce yeast cells where the desaturase worked just as it did in the chloroplasts, proving that the head group was the key to the location-dependent change in function.

Now the Brookhaven scientists, together with collaborator Thomas Girke of the University of California at Riverside, are conducting studies to see how widespread this enzyme function-shifting phenomenon might be. Using computer algorithms, they've analyzed the entire Arabidopsis genome for variations in "address signals." Of the approximately 6,000 Arabidopsis gene families, they've identified 239 that produce enzymes that are sent to different locations within cells. Whether these variants have different functions remains to be seen. "It's a new area that needs further exploration," Shanklin says.

The scientists will also explore the possibility of adding or removing address signals to/from known enzymes to see if they can change their locations and functions. "We are already using the chloroplast desaturase, without its address signal, to make a healthier, less saturated, plant oil in Arabidopsis," said Shanklin - an approach, he says, that might one day be useful in producing healthier crop plants.

This research was funded by the Office of Basic Energy Sciences within the U.S. Department of Energy_s Office of Science, The Dow Chemical Company and Dow AgroSciences LLC, and the German Science Foundation.

2004-196  |  Media & Communications Office

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