"Designer Enzyme" Illuminates Subtle Difference Between Plant And Animal Protein

BUFFALO, N.Y. -- University at Buffalo biochemists have taken an enzyme from a bacterium found in plants and, to their surprise, genetically engineered a mutant that has an important property normally found in the animal version of the enzyme: It requires zinc instead of magnesium to function properly.

The enzyme was inserted back into the bacterium, which was introduced into soybean seedlings. The plants containing the mutant grew normally, demonstrating that the animal version could function in plants.

The research was published in the August 25 issue of the Journal of Biological Chemistry.

According to the co-authors, the research has important applications for protein engineering and research on agriculture and the environment. It also provides insight into the differences between plants and animals.

"It was very surprising that we were able to alter a fundamental property by making only a small change in the enzyme," said Mark R. O'Brian, Ph.D., associate professor of biochemistry at UB and lead author on the paper. UB postdoctoral researcher Sarita Chauhan, Ph.D., was co-author.

"It was equally surprising that the enzyme functions in bacterial cells when they are put back into the plant," he said.

In making the altered enzyme, the researchers have demonstrated the potential for enzyme engineering, where scientists can design new enzymes with special properties.

O'Brian added that what may be even more illuminating is what the research contributes to knowledge about the scientific differences between plants and animals.

"From an evolutionary perspective, this work emphasizes that small changes in a protein -- in this case, just a few amino acids -- can have very significant effects that may allow the organism to adapt," he said.

The UB researchers started with an enzyme called ALAD, (aminolevulinic acid dehydratase), which is involved in the synthesis of hemes and chlorophylls. These colorful compounds give blood their red color and make plants green. The ALAD used in the study was from the bacterium Bradyrhizobium japonicum.

The scientists were interested in this enzyme because of the role of Bradyrhizobium japonicum in nitrogen fixation, a process that converts atmospheric nitrogen into a form that allows the plants with which the bacteria associate to thrive in soils deficient in the nutrient. The Bradyrhizobium japonicum bacteria form nodules on the roots of soybean plants, which allow them to grow under conditions where other plants, such as corn or wheat, cannot grow.

"It takes a lot of cellular energy for bacteria to fix nitrogen," explained O'Brian. "That's why we're interested in heme proteins, because they allow the bacteria to make energy."

Previous work he had published in Science suggested that the synthesis of heme might be regulated at the level of ALAD.

"We suspected that ALAD production was a regulating step in the pathway, that is, the enzyme controls what quantity of heme is produced, and that's probably true," O'Brian said.

The difference between the plant and animal versions of the ALAD enzymes lies in the metals they need to function properly: In plants, the enzyme requires magnesium, while in animals, as well as most bacteria, the enzyme requires zinc.

This particular ALAD is somewhat different in that even though it is from a bacterium, it requires magnesium in order to function.

By changing just four amino acids in this 353-amino acid protein, the UB researchers developed a mutant enzyme that requires zinc to function.

In addition to successfully making this switch, the UB researchers went one important step further. They inserted the mutant enzyme back into the bacterial cell, which they introduced into soybean seedlings.

"The whole plants were able to fix nitrogen in their nodules and grow normally with the mutant enzyme," O'Brian said.

"We still don't know whether the engineered ALAD would function in leaves to make chlorophyll, but it clearly works in nodules," he noted.

This means that an animal version of ALAD can function in plant tissue.

"It may be difficult to believe, but at the molecular level, plants and animals are more similar than they are different," said O'Brian. "That is why pesticides used to kill plants can be potentially harmful to people and animals. Therefore, defining specific molecular differences between plants and animals may aid the development of safer, more effective pesticides."

The knowledge also can be applied in phytoremediation, a new environmental technique in which plants are used to clean up sites where soils have been contaminated with toxic levels of metals, O'Brian said.

In addition, the work may contribute toward understanding how proteins with desirable properties can be engineered for medical, agricultural and commercial uses.