March 2, 2000
UPTON, NY - A team of scientists working at the U.S. Department of Energy's Brookhaven National Laboratory has determined the atomic structure of a key enzyme that performs several important chemical jobs in the body, including synthesizing estrogen and detoxifying chemicals as they enter the body. In addition, the enzyme may have medical and commercial applications in cancer therapy, in reducing pollution in industrial waste streams, and in manufacturing epoxies.
Specifically, the researchers obtained three-dimensional images of the protein called cytochrome P450cam from the bacterium Pseudomonas putida. Part of what makes this work so interesting is that they have produced a molecular movie, visualizing five critical steps in one of the enzyme's typical chemical reactions.
The researchers used an intense x-ray beam from one of the facilities, or "beam lines" of Brookhaven's National Synchrotron Light Source (NSLS) to perform the study. In the reaction, the enzyme oxidized, or added oxygen atoms to a hydrocarbon molecule. The research collaboration referred to the enzyme as "the biological equivalent of a blowtorch" because it is so highly reactive with so many organic compounds. Their new data are helpful in understanding how this key enzyme functions.
Robert Sweet, a Brookhaven Lab scientist who participated in the experiment and designed the beam line on which it was done, said, "For the first time, we witnessed this powerful enzyme as it oxidized hydrocarbons - a reaction that can be exploited to clean up pollutants or synthesize new compounds."
The results of this nine-year x-ray crystallography study at Brookhaven's National Synchrotron Light Source (NSLS) are reported in the March 3 issue of the journal Science. The collaboration includes researchers from Brookhaven Lab, the Max Planck Institute for Molecular Physiology, Los Alamos National Laboratory, the Center for Advanced Biotechnology and Medicine in New Jersey, the University of Illinois, and Brandeis University.
This research is the first step in analyzing a class of enzymes that may be used in cancer therapy by shutting down estrogen synthesis in the body. In a completely different application, genetically engineered P450cam enzymes may be used to eat up toxic chemicals, such as PCBs and trichloroethylene, in chemical manufacturing waste streams, thus preventing groundwater pollution. Finally, they may be used in an environmentally friendly, energy-efficient way to make epoxies.
Sweet explained the experiment, saying, "We arranged to take the enzyme through its catalytic cycle, and we trapped it during various crucial steps by freezing it with liquid nitrogen. We stimulated it to go through the cycle by using low-energy x-rays and by a sequence of thawing and refreezing."
The researchers grew crystals of cytochrome P450, and combined it with camphor, an ingredient of some mothballs; and several other chemicals. The compound then was exposed to synchrotron x-rays, which added electrons to the mixture, causing a chemical reaction that is the hallmark of the enzyme: It splits molecular oxygen into two atoms and inserts one of the atoms into a hydrocarbon - in this case, into camphor. While the researchers studied an enzyme from a specific bacterium, this reaction is thought to be common to all P450 enzymes.
The work on this project was performed at the structural biology facilities at Brookhaven's NSLS, which are currently the most productive in the world. The facility, consisting of seven beam lines with state-of-the-art instrumentation for structural biology studies, is a resource for scientists from universities, industry and other laboratories from all over the world.
The U.S. Department of Energy's Brookhaven National Laboratory creates and operates major facilities available to university, industrial and government personnel for basic and applied research in the physical, biomedical and environmental sciences and in selected energy technologies. The Laboratory is operated by Brookhaven Science Associates, a not-for-profit research management company, under contract with the U.S. Department of Energy.