Parasite’s enzyme structure helps address a public health issue

By revealing the architecture of an essential enzyme in a parasite, Dartmouth researchers are helping address a public health issue.

Researchers in the laboratory of Amy Anderson, Assistant Professor of Chemistry, have unveiled the structure of an enzyme called dihydrofolate reductase-thymidylate synthase, also known as DHFR-TS, from a waterborne parasite called Cryptosporidium hominis. Knowing the chemical structure of the enzyme will help researchers design highly targeted drugs to combat the parasite, which needs this enzyme to reproduce.

“We wanted to know how DHFR-TS is assembled and how it works,” says Anderson. “Then we’ll know how to disable it and kill the parasite.”

Anderson, along with Robert O’Neil, a senior researcher and the lead author of the study, Ryan Lilien, an M.D./Ph.D. graduate student at Dartmouth, Bruce Donald, Professor of Computer Science, and Robert Stroud, Biochemistry and Biophysics Professor at Univ. of Calif. at San Francisco, have solved the puzzle of DHFR-TS by revealing its chemical architecture.

Their results were electronically published on October 9 in an online issue of the Journal of Biological Chemistry, and the paper appeared in the print edition of the journal on December 26, 2003. The study was also rated as “exceptional” and a “hidden jewel in microbiology” by the Faculty of 1000, a group of researchers who rate published articles in the life sciences each month.

The Centers for Disease Control and Prevention (CDC) have been watching Cryptosporidium and tracking its impact on human populations, where it spreads easily and quickly, for more than 20 years. While healthy people stricken with this parasite usually recover on their own, it can be deadly for children, elderly people and those whose immune systems are compromised, like people with HIV/AIDS or patients undergoing chemotherapy. According to the CDC Web site, Cryptosporidium is often found in public water supplies in the U.S. and cannot be easily filtered out or killed by traditional treatments like chlorine. Currently, there is no cure, and available medicine only eases the symptoms.

The study helps better define the evolution of this protozoan family that includes Plasmodium, which causes malaria, and Toxoplasma, which induces toxoplasmosis, a disease that can lead to central nervous system disorders. Knowing how this one enzyme is assembled will help researchers better understand related parasites, Anderson says.

“By using the structure of many protozoan DHFR-TS enzymes, we’ve been able to place a number of protozoa in distinct evolutionary families. This is the first time that this enzyme has been used to do this,” says Anderson. “It’s an important distinction that helps classify the protozoa, and helps us design more effective drugs to combat them.”

To discover DHFR-TS’s nuts and bolts, Anderson and her team used a process called “protein crystallography.” The process involves taking DNA from Cryptosporidium and cloning it in the fast-growing bacteria E. coli to harvest large amounts of the target enzyme. Researchers then break E. coli open to release all of its proteins. All of the proteins are mixed with beads, or tags, which “grab” just the DHFR-TS enzyme.

Once DHFR-TS has been isolated, it’s collected in a tube, concentrated and crystallized. The crystal, which is an ordered array of enzyme molecules, is subjected to a powerful X-ray beam. Diffracted X-rays emerge and are imprinted on a film. The researchers use mathematical algorithms to interpret the X-ray data, which eventually reveal the structure of the protein.

“We can place every atom in the protein, and we can chart their interactions,” says Anderson. “We learn how the protein is put together and which atoms bond to one another. It’s important to learn the structure of a protein to figure out how it works.”

Researchers in Anderson’s lab also work on the same enzyme model for Toxoplasma, cousin to Cryptosporidium. The advantage, Anderson explains, is that by solving this enzyme’s structure for both Cryptosporidium and Toxoplasma, they can better predict how it will look in their other family members, like Plasmodium, the malaria bug.

Anderson’s team is also working on “structure-based drug design” to carefully design drugs to interact with the specific enzyme to influence how the enzyme will function.

“We want to prevent DHFR-TS from doing its job in the parasitic organism without stopping the human enzyme that looks similar,” says Anderson. “DHFR and TS have similar functions in humans: they are critical to DNA replication.”

The National Institutes of Health funded this study.