Scientists at the U.S. Department of Energy's Brookhaven National Laboratory, the New York Structural Biology Center, and SGX Pharmaceuticals, Inc., have determined the atomic crystal structure and functional mechanism of an enzyme essential for eliminating unwanted, non-nutritional compounds such as drugs, industrial chemicals, and toxic compounds from the body. The detailed mechanism of action will help scientists understand how these compounds are eliminated and what goes wrong in cases where normal metabolism fails. The research is published online in June, 2006, by the Proceedings of the National Academy of Sciences.
According to Brookhaven biologists Eswaramoorthy Subramaniam, the lead author, and Subramanyam Swaminathan, who led the research, most non-nutritional, foreign substances such as drugs and industrial chemicals are insoluble in water. The body uses two main groups of enzymes -- flavin-containing monooxygenases (FMOs) and cytochrome P450s -- to convert these compounds to soluble forms that can be easily excreted.
"For FMOs, the end result -- that an oxygen atom gets added to make these compounds soluble -- is simple," Swaminathan says, "but the reactions require additional participants, or cofactors." In order to understand the molecular mechanism, the scientists used high-intensity x-ray beams at the National Synchrotron Light Source (NSLS) to identify the positions of individual atoms and produce crystal structures of the enzyme, the enzyme plus its cofactor, and the enzyme plus the cofactor plus the compound to be oxidized (the substrate).
"These crystal structures give step-by-step snapshots of different stages of the catalytic action," Swaminathan says, "and reveal a mechanism that is different from what had been known about this process."
Previously, it had been believed that all the "players" -- the enzyme, cofactor, and substrate -- came together at a particular time to perform the function of transferring an oxygen atom from the enzyme to the substrate. "Our finding shows that the substrate and cofactor are binding to the enzyme alternately, not together," Swaminathan says.
First, the cofactor (known as NADPH) binds to a molecule known as FAD, which is a coenzyme attached to the FMO, and transfers a hydride ion to it. That makes the FAD group capable of accepting molecular oxygen. Then, when the substrate arrives, the cofactor leaves so that the substrate can bind to the same site on the FAD group. At this moment an oxygen atom from molecular oxygen is attached to the substrate, and the hydride ion obtained from the cofactor combines with the other oxygen atom to form a water molecule, which is released. Once the substrate is oxygenated, it leaves the enzyme and the cofactor binds again.
"With this back-and-forth, alternating binding, the process repeats over and over for continuous turnover of the product," Swaminathan says.
The details of this process may help scientists understand what happens in cases where compounds are not properly metabolized, and possibly develop corrective measures.
One example is a condition called trimethylaminuria, also known as "fish odor syndrome," which results from defective FMOs. Affected individuals are unable to oxygenate trimethylamine, a byproduct of protein digestion released by bacteria living in the gut. People with the disorder release trimethylamine through breath, sweat, and urine, producing a fish-like odor that can be embarrassing and result in psychological effects such as withdrawal and depression.
People with defective FMOs might also suffer additional side effects from drugs, industrial compounds, or other chemicals.
This research was funded by the National Institutes of Health and was conducted at the National Synchrotron Light Source, supported by the Office of Basic Energy Sciences within the U.S. Department of Energy's Office of Science.
Upon publication, the PNAS paper will be available online at: http://www.pnas.org/cgi/doi/10.1073/pnas.0602398103
One of ten national laboratories overseen and funded primarily by the Office of Science of the U.S. Department of Energy (DOE), Brookhaven National Laboratory conducts research in the physical, biomedical, and environmental sciences, as well as in energy technologies and national security. Brookhaven Lab also builds and operates major scientific facilities available to university, industry and government researchers. Brookhaven is operated and managed for DOE's Office of Science by Brookhaven Science Associates, a limited-liability company founded by the Research Foundation of State University of New York on behalf of Stony Brook University, the largest academic user of Laboratory facilities, and Battelle, a nonprofit, applied science and technology organization. Visit Brookhaven Lab's electronic newsroom for links, news archives, graphics, and more: http://www.bnl.gov/newsroom
Note to local editors: Subramanyam Swaminathan is a resident of Coram, New York.
Source: DOE/Brookhaven National Laboratory, June 2006