Naturopathic Medicine, Agricultural Engineering, And Biofuels

The Quest To Understand How Plants Produce An Amino Acid Essential For Human Survival

By Charla Lambert

In Manhattan, a woman with a family history of heart disease drinks a glass of merlot with her weekday dinners.

In Portland, Ore., a man switches his morning beverage from coffee to green tea.

Meanwhile, a woman in Tallahassee, Fla. takes daily doses of soy-based estrogen as an alternative to conventional hormone-replacement therapies.

The active ingredients in red wine, green tea, and soy-based products have all been shown to have positive effects on human health. While certain teas have antioxidant properties, for example, red wine may lower a person's risk of cardiovascular disease. Remarkably, the active ingredients are part of a single family of compounds: they are all products of one biochemical pathway that begins with the amino acid phenylalanine, or Phe for short.

Bacteria and plants can make phenylalanine, and thus its derivative compounds, on their own. Humans, however, cannot. While phenylalanine is a necessary nutrient for humans and its derivatives quite beneficial, we must rely on either bacteria or plants to make it for us.

For decades, scientists have struggled to understand how plants synthesize phenylalanine. Because it is essential for human health, the biochemical reactions that create phenylalanine are crucial to the survival of humans as well as plants. "If these don't exist, we don't exist. It's as simple as that,” says Norman Lewis, a biochemist at Washington State University (WSU) in Pullman. Now, Lewis and his colleagues have made progress in unraveling a key step in the mystery using the small flowering plant Arabidposis thaliana. The work was published in a recent issue of the Journal of Biological Chemistry.

A complete understanding of phenylalanine biosynthesis could eventually allow scientists to engineer medicinal plants, increasing yields of essential nutrients and active compounds without over-harvesting. "Plant extracts have been used since ancient times to treat many kinds of diseases or physiological maladies, although the practitioners never knew the mechanisms of action of their medicines,” says Filippos Ververidis in a recent review of Phe-derived natural products. Ververidis, a plant biochemist in Heraklion, Greece, believes the present-day challenge is to understand the biochemistry of such plant extracts and develop alternative ways of producing them.

In addition to medicinal benefits, an understanding of phenylalanine biosynthesis has technological applications that range from frivolous to practical. At one end of the spectrum, Phe-derived products control flower pigmentation. Perhaps you're a die-hard New York Giants fan and you'd like to decorate your living room with blue and white flowers, in honor of the Giants' 2008 Super Bowl victory. However, the perfect deep blue flower that matches the face paint you sported during the big game doesn't seem to exist. One day in the future, plant scientists may be able to help you out.

At the other end of the spectrum, Phe-derived products are also anti-microbial agents and insect repellants. These defensive compounds naturally protect plants from herbivores that feast on their stems and leaves. With a complete biochemical understanding of the ways in which plants synthesize and use phenylalanine, agricultural scientists could one day manipulate crops so that harsh chemical insecticides are no longer needed.

Norman Lewis came to study phenylalanine and its derivatives, collectively referred to as phenylalanine metabolism, through his interest in yet another Phe-derived product called lignin. Similar to the human skeleton, lignins provide structural rigidity in trees and woody plants; they are also necessary for maintaining the integrity of plant cell walls. Over the past two decades, Lewis has investigated the ways in which plants assemble lignins, with the hope of understanding how scientists might efficiently break them down. He explains: "The main biotechnological challenge is to take the genetic information leading to these lignified woody tissues and see if these vascular plants can be changed so that their structural properties remain the same, but are easier to break down and/or remove.” Manipulating plants in this way would make them more cost-effective to process into consumer goods such as paper and fuel. This area of research has the potential to dramatically affect the state of energy supplies and shortages in the U.S. "We envisage that manipulating structural properties of plants in ways such as this will aid in production of biofuels, such as ethanol,” says Lewis.

For his most recent study, Lewis set out to investigate a very specific question: Which of two candidate enzymes do plants utilize on the pathway to phenylalanine? Uncovering the enzyme also uncovers the biochemical mechanism underlying that step in the pathway, providing scientists with much-needed information in the quest to fully understand phenylalanine metabolism. Indeed, many research groups have studied this question since the 1980s, but results have proven inconclusive. The difficulty lies in the fact that the two enzymes, arogenate dehydratase (ADT) and prephenate dehydratase (PDT), are inherently unstable. Like chocolate candy on a hot summer day, the enzymes deteriorate and fall apart easily in laboratory experiments.

Lewis and his colleagues at WSU and the University of Western Ontario approached the question from a radically different perspective. Rather than starting from a plant and isolating proteins, they started from DNA sequences within the Arabidopsis thaliana genome. With the sequences in hand, the scientists were able to express the genes in cell culture and examine their functions directly to determine whether they most resemble ADT or PDT.

The six genes that served as a starting point for the experiments are similar at the DNA-sequence level to bacterial genes encoding the PDT enzyme. This sequence similarity provides a reasonable yet crude hypothesis for the gene functions within Arabidopsis: they are likely enzymes that also act like PDT.

The process of generating functional hypotheses based on sequence similarity is a common practice in the emerging field of genomic science, especially for organisms like Arabidopsis that have a large number of uncharacterized genes. Sequence similarity does not always translate to functional similarity, however. It turns out that all six genes Lewis and his colleagues examined exhibit behavior more similar to ADT than to PDT. Three of the genes show low levels of PDT activity while the other three show nearly none. At the same time, all six produce high levels of derivative compounds associated with ADT.

For now, the researchers have characterized all six genes as putative ADT enzymes. They are currently investigating when and where the genes are turned on in Arabidopsis, to piece together the complete puzzle of phenylalanine metabolism in the plant.

Charla Lambert is a graduate student in genome sciences at the University of Washington.

Images:

The flowering mustard Arabidopsis thaliana is a model organism used by many researchers in plant genetics. Photo: The Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org).