Newswise — Researchers at Washington State University have identified the plant enzyme responsible for making phenylalanine, an amino acid that is an essential nutrient for humans. Phenylalanine is also an essential starting material for making flower pigments, substances that protect against UV irradiation from the sun, and wood.

The research team, led by Norman Lewis of WSU's Institute for Biological Chemistry, has cloned six genes coding for different forms of the enzyme arogenate dehydratase (ADT), which converts a compound called arogenate into phenylalanine. Their work will appear in the Oct. 19 issue of the Journal of Biological Chemistry.

A description of phenylalanine's fate underscores its central role in terrestrial plant life and the importance of the enzymatic reaction that produces it.

Phenylalanine is converted into phenolic compounds that are the building blocks of many of the plant world's most distinctive and important substances, including the pigments in flower petals and chemicals that protect leaves, stems and bark from ultraviolet radiation. Perhaps the best-known end product of phenols is the one that allows trees to stand upright.

Compounds made from phenylalanine are assembled into the bio-polymer lignin, which is a major structural component of wood—and the second-most abundant organic material in nature, said Lewis. (Cellulose, another plant polymer, is first.)

"In woody plants, more than 30 percent of the carbon has at one point been [in] phenylalanine," said Lewis.

Scientists have puzzled for decades over how plants make phenylalanine. Two possible routes had been identified through work with bacteria, but evidence for either route in plants has been sketchy. Traditional methods of trying to isolate the two enzymes of interest (ADT and prephenate dehydratase, or PDT) had been unsuccessful, because the enzymes are unstable and fall apart during or soon after purification. The substrate for ADT, arogenate, is also quite unstable and does not accumulate in plants to any great degree.

So Lewis's team came at the problem from a different direction. Instead of purifying the protein first and then looking for the gene that codes for it, they took advantage of the information made available by recent advances in gene sequences. They used the new technique of data mining to search a known plant genome sequence for genes that were likely to code for the enzymes they were looking for. The genome was that of the small mustard Arabidopsis, commonly used in experiments on plant physiology.

The researchers found six gene sequences that were similar to bacterial PDT. They cloned the six genes and inserted each into E. coli bacteria, which produced the proteins the genes coded for. Lewis' team stabilized the proteins by attaching them to another protein that allowed them to function properly and remain intact through the course of subsequent experiments.

The resulting proteins all proved to be forms of ADT, efficiently converting arogenate to the end product, phenylalanine.

Lewis said his group is now working to determine which of the six genes are turned on in various tissues and at different developmental stages, to get a clearer picture of phenylalanine production and use within the plant.

Phenylalanine is more than a precursor to other important compounds. Since it is an amino acid, it is used as a building block itself in the production of proteins. That happens in animals as well as in plants; humans and other mammals, however, can't produce phenylalanine. We obtain it by breaking down proteins in the food we eat—either plant material, or the meat of animals that ate plants.

Lewis said our reliance on plants to make phenylalanine means the reactions that produce it are as crucial to our survival as they are to that of plants.

"If these don't exist, we don't exist. It's as simple as that," he said.

To read the full paper, visit http://www.jbc.org/cgi/reprint/282/42/30827 (PDF).

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Journal of Biological Chemistry (19-Oct-2007)