HOUGHTON,MI -- Researchers at Michigan Technological University are studying an enzyme that is being used to help clean up the environment and has great potential to be part of the solution to the global problem of excess nitrate and related nitrogen nutrients in water sources.
The enzyme--nitrate reductase--comes from plants where it plays a central role in nitrate acquisition and is essential for feeding people and animals.
Dr. Wilbur H. Campbell, professor of biochemistry and molecular biology and director of Michigan Tech's Phytotechnology Research Center, heads a team of undergraduate and graduate students and a post-doctoral fellow whose aim is to determine the structure and function of nitrate reductase. They want to create a 3-D model of the enzyme to gain understanding of how it works.
Campbell's research is currently being funded by a 3 year, $300,000 grant from the National Science Foundation. But in fact, he has been doing basic research on nitrate reductase for more than 20 years with grants from NSF, the US Dept of Agriculture and the State of Michigan's Research Excellence Fund.
Nitrate reductase is in virtually every plant on earth and is a very efficient enzyme with an important job in nitrogen metabolism in plants, according to Campbell. The problem is--plants don't need much nitrate reductase to get their job done and so nature doesn't provide enough of the enzyme for scientists to do the studies they need for determining just how the enzyme functions and what its full potential may be.
Interestingly, nitrate reductase has a role in environmental biotechnology where it is being used as a nitrate testing method. This new commercial method for testing for nitrate in water has been developed at a small biotechnology company in Lake Linden, Michigan, called the Nitrate Elimination Co., Inc. The company was started by Campbell and his wife Ellen in 1993 to produce nitrate reductase using a process they licensed from Michigan Tech. Research and development of nitrate reductase-based products has been funded by Small Business Innovation Research grants from the EPA , USDA and the Michigan Department of Commerce.
Environmental testing for nitrate is often done with methods based on heavy metals like cadmium and zinc. Nitrate reductase-based nitrate testing replaces these older methods, which have potential to harm the environment, with an environmentally friendly method that is safer for the person doing the test and at the same time can be more accurate, according to Campbell. In addition, the Campbells' small company is doing research and development on an enzyme-based electronic detector for real-time continuous monitoring of nitrate in water and a method for removing nitrate from water using enzymes. The nitrate removal method could be used in homes and businesses with nitrate polluted water, which would make it fit to drink or use for the production of foods and beverages.
"We don't have a way to make enough of it to modify the enzyme's protein and change its amino acids," says Campbell, " and those are the kind of studies we need to do to understand how the protein part in nitrate reductase functions with its cofactors that include a flavin (vitamin b-2), heme iron (like in hemoglobin) and molybdopterin, which is also an essential cofactor for human health. All these components work together to catalyze the conversion of nitrate to nitrite but we don't know how it really works on the molecular level. That's what we want to find out."
In an attempt to overcome the scarcity factor, Campbell and other research groups like his are working to develop a process that will result in "recombinant" expression of the nitrate reductase gene and will provide scientists with the large amounts of the material they need for experiments.
"It's been a step-by-step process," explains Campbell. "When we first started recombinant expression of nitrate reductase, we couldn't produce an entire enzyme--now we can." The Michigan Tech Professor has worked with Swedish crystallographers who crystallized the first part of the enzyme, which was produced in a bacterium in 1989. This allowed them to determine the 3-dimensional structure of this fragment of nitrate reductase, which houses the flavin in the complete enzyme. Now they can "see" it through the use of a computerized model.
"We're studying the structure of the enzyme with the goal of understanding how it catalyzes the nitrate reduction process," says Campbell. "In the next couple of years we hope to get extensive information about crystal structure that will help us solve the structure/function mystery. Ultimately we need to be able to produce large quantities of enzyme to use in these experiments."
Campbell says the recombinant process enables scientists to clone a gene from a plant, put it into a vector that "allows us to manipulate the cloned gene and place it into a yeast where a promoter is 'triggered' by methanol and allows us to control when the gene is expressed. The end result is an enzyme that is very much like the natural nitrate reductase we started out with. This gives us capacity to produce the qualities of the enzyme we need for research." He also says this is the first step toward making nitrate reductase commercially available on a very large scale, which will make it useful to industry.
Successful completion of the Michigan Tech studies will lead to a complete and detailed picture of nitrate reductase structure and function and will guide future studies of the enzyme, according to Campbell. Overall, he feels his research program is a good example of how basic research, even into an esoteric plant enzyme, has a potential payoff for society in terms of a higher quality of life and tends to help prove the value of basic research as a tool for improving America's economic competitiveness in the new global marketplace.
Materials provided by Michigan Technological University. Note: Content may be edited for style and length.
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