On Research:
Unlocking Enzymes'
Secrets May Be
Fertile Idea

The enzymes--nitrogenase and hydrogenase--are present in bacteria called Rhizobia, which grow on the roots of plants such as soybeans and other legumes, enabling those crops to take nitrogen directly from the air. The bacteria "fix" nitrogen into the soil and then nitrogenase makes ammonia fertilizer by combining hydrogen with the nitrogen, saving U.S. farmers about $8 billion in fertilizer costs annually.

Most crops, such as corn and wheat, cannot make their own fertilizer. They must be grown with nitrogen-based fertilizers, which are costly and difficult to produce.

"You have to physically combine hydrogen and nitrogen at very high pressure," said Hopkins Biology Professor Robert Maier.

Another economic factor is the price of nitrogen, which is subject to market fluctuations and has risen in recent years. Farmers in the United States grow about 80 million acres of corn each year. In 1995, the most recent year for which statistics are available, the cost of fertilizing corn took an astounding leap, from $6 to $14 per acre. The same year, fertilizer costs for wheat rose 15 percent.

"There was a huge increase in the cost of nitrogen in 1995," said Chris McGath, an economist with the U.S. Department of Agriculture.

The market price for nitrogen hit an all-time high, underscoring the need for research that could lead one day to alternatives to nitrogen-based fertilizers.

Perhaps Rhizobia represent one solution to the problem, said Maier, who is acting chair of the Biology Department. Scientists have been trying to learn how to incorporate Rhizobia into food crops and to improve its efficiency, even in plants that already harbor the bacteria.

That's because some Rhizobia are inherently more efficient than others. When nitrogenase makes ammonia, hydrogen is released as a wasted by-product. In most cases, the gas is given off, rising invisibly from the fields. But some forms of Rhizobia are equipped with hydrogenase, using that enzyme to turn hydrogen into energy and making the plants more efficient. Those species of Rhizobia increase crop yields by as much as 8 percent, Maier said.

In 1986 Maier discovered essentially how hydrogenase works; he learned that it contains a large amount of the metal nickel, which it uses to attract hydrogen. He then spliced DNA from Rhizobium into Escherichia coli bacterium. As the E. coli expressed the Rhizobium gene, it produced large quantities of an unusual nickel-storage protein, which scientists now know is a vital component of hydrogenase.

He was astonished to learn how much of the metal was stored in the protein.

"It binds 18 nickel atoms per molecule," Maier said. "The previous highest nickel-binding protein was four, four nickels per molecule. This had 18 nickels per molecule."

The protein, which Maier named nickelin, provides a ready supply of nickel for the bacterium.

"Then when the organism needs to make this hydrogenase, it uses the nickel," said Maier, who detailed some of his most recent findings in two scientific papers published this summer in the Journal of Bacteriology.

But how exactly does the nickel get transferred to the enzyme?

"We are trying to figure out how it does that," he said. "It's getting complicated. I've worked on hydrogenase for 18 years, and I really want to determine how this occurs."

In addition to its possible agricultural value, nickelin's ability to store metal makes the protein potentially useful for cleaning up the environment. Scientists are exploring whether it might be used in plants genetically designed to sop up environmental contaminants.

Maier has identified the 22 genes in Rhizobia that regulate the use of hydrogenase. Biologists want to insert the genes into the DNA of bacteria that grow on crops other than soybeans, making those plants more productive in nitrogen-poor soils.

But they also are trying to make plants more productive by increasing the efficiency of the nitrogenase itself.

"There is no doubt that, to help solve world hunger and to make an impact, this Rhizobium must be improved," Maier said.

Maier and scientists in his lab recently have made strides toward reaching that goal, by studying another bacterium called Azotobactor, which also fixes nitrogen from the atmosphere.

Azotobactor uses two biochemical mechanisms that protect its nitrogenase from being damaged from exposure to oxygen. In Rhizobium, oxygen atoms pluck away electrons from nitrogenase, inflicting irreversible damage that hinders the enzyme's efficiency. That's because Rhizobium lacks the protective mechanisms that Azotobactor uses to safeguard its nitrogenase from oxidation damage.

"If it had these mechanisms, I think nitrogenase would be improved," said Maier, who has discovered which genes regulate the mechanisms and hopes to find a way to incorporate them into the DNA of Rhizobium.