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Professor Amy Anderson Enzyme Architecture Written by Susan Knapp

 

Amy Anderson is an assistant professor of chemistry.


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If researchers can figure out how the enzyme that allows the deadly Cryptosporidium parasite to reproduce, they can then find ways to disable it.

About ten years ago, an estimated 403,000 people in the Milwaukee area were stricken with severe intestinal distress. The culprit fingered in this crisis was a one-celled parasite called Cryptosporidium parvum. The Centers for Disease Control and Prevention have been watching this protozoan and tracking its impact on human populations for more than twenty years. In fact, the CDC lists Cryptosporidium as a Category B disease or agent, which falls in the second highest group of potential biological threats. To put that in perspective, Category A threats include anthrax and smallpox.

The atomic structure of an enzyme drug target from the water-borne pathogen, Cryptosporidium parvum.
The atomic structure of an enzyme drug target from the water-borne pathogen, Cryptosporidium parvum.

The atomic structure of an enzyme drug target from the water-borne pathogen, Cryptosporidium parvum. Drugs bound to the enzyme (dark blue ball-and-stick molecules) are being modified to increase their binding ability and selectivity. (Illustrations courtesy of the Anderson lab)

Cryptosporidium is a nasty creature. It spreads easily and quickly. 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. Cryptosporidium is often found in public water supplies in the U.S. It cannot be easily filtered out or killed by traditional treatments like chlorine. Right now, there are no effective treatments. Medicine only eases the symptoms.

Amy Anderson wants to know what makes Crypto-sporidium tick in order to design drugs that will kill it. Her laboratory is studying an enzyme that allows Cryptosporidium to reproduce. Called dihydrofolate reductase-thymidylate synthase, this enzyme is also known as DHFR-TS. Cryptosporidium has thousands of enzymes, but DHFR-TS is a particularly important one because Cryptosporidium needs this enzyme to live.

"We want to know how DHFR-TS is assembled and how it works," says Anderson. "Then we'll know how to knock it out to kill the parasite."

Enzymes are unique proteins, and proteins, produced by genes, carry out all cellular functions. Now that the human genome and others have been mapped, the next great scientific frontier will be learning how genes work, what proteins the genes influence, and how the proteins work.

Anderson and her colleagues have solved the puzzle of DHFR-TS by revealing its chemical architecture. Knowing how this enzyme is built allows them to better determine how to disable it. If the enzyme is deactivated, the parasite can't reproduce and the illness is, in effect, cured.

There have been some challenges along the way. This particular enzyme, in addition to its vital role in reproduction, has some interesting properties.

"In people, these enzymes are separate. In bacteria, these enzymes are separate. In protozoa, they are fused," says Anderson. "It's so exciting because this is the first DHFR-TS structure to be solved in this family of protozoa."

Cryptosporidium belongs to a protozoan family that also 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 built will help researchers better understand related parasites, Anderson says.

"We're learning a huge amount about the reasons that Cryptosporidium is resistant to current drugs, how to design new drugs, and how to apply this model to the rest of the family," says Anderson.

An NRPS enzyme being redesigned by Amy Anderson and Bruce Donald

An NRPS enzyme being redesigned by Amy Anderson and Bruce Donald, professor of computer science, using computer modeling techniques. These newly designed enzymes might some day give rise to new generations of antibiotics. (Illustration courtesy of the Anderson lab)

To discover DHFR-TS's nuts and bolts, Anderson and her team used a process known as protein crystallography. The procedure involves taking DNA from Cryptosporidium and cloning it in the fast-growing bacteria E. coli to harvest the target enzyme. Researchers then break E. coli open to release all of its proteins which are then mixed with beads, or tags, that "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.

"We think this will lead to a novel model for malaria," says Anderson. "All of the drugs that have been aimed at this enzyme in malaria are based on an old model. If we can better predict what malaria looks like and how it acts -and find out why malaria is so resistant to so many drugs-it would make a huge impact on the millions of people who suffer and die from this disease."

At the same time that Anderson's team was occupied by the crystal structure pursuit, they also tackled structure- based drug design. Carefully designing drugs to interact with any specific enzyme profoundly influences how the enzyme will function.

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

The drug design-testing efforts are already showing that some new Toxoplasma drugs are seven hundredfold selective for the parasite over the human form of the enzyme.

"Specificity is what we're shooting for," she says.

Sidebar

"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."

 
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