Based on these new insights into the structure of the enzyme, called DHPS, the St. Jude team has also developed a new molecule that appears likely to be able to block the enzyme's activity without triggering resistance.
DHPS normally combines the molecules DHPP and pABA during part of a biochemical pathway that produces folate, a nutrient these bacteria need to survive. Sulfa drugs are antibiotics that block pABA from binding to the enzyme, and thus block folate production.
The St. Jude findings give important clues to how the enzyme binds to DHPP and pABA. They also provide a blueprint of the enzyme that researchers can use to design more effective antibiotics against the bacterium, Bacillus anthracis. Such information is especially valuable because the anthrax bacterium is widely regarded as a potential bioterrorism weapon. The finding could also be the basis for a broad-spectrum antibiotic to treat a variety of other infections that are becoming resistant to these drugs.
The team made these discoveries by creating images of the molecular structure of DHPS using X-ray crystallography. The team bombarded crystals of the enzyme with X-rays, then used the patterns formed by the diffraction of the beams off the crystals to create computer-generated, three-dimensional images of the enzyme shape. The researchers also used this technique to make images of the enzyme bound to the two molecules that the enzyme chemically combines with to make folic acid.
Based on these images of the enzyme and its interaction with other molecules, the investigators discovered how it manipulates pABA and DHPP. In addition, the images disclosed a potentially new antibiotic target within the enzyme structure that would be much less likely to develop resistance to an antibiotic, the researchers say.
"Until now, no one had produced images of sulfa drugs binding to DHPS," said Stephen White, Ph.D., chairman of the St. Jude Structural Biology department. "Without that kind of visual information it's impossible to fully understand how the enzyme works, how the sulfa drugs interfere with DHPS and what kind of changes in the structure of DHPS make this enzyme resistant to sulfa drugs."
White is senior author of the Structure report. The St. Jude investigators created images of the loops and helical ribbons of protein making up this DHPS. In addition, they created images showing how certain small enzyme mutations allow it to alter its structure, blocking the attachment of sulfa drugs. The image showed that while this slight change in the shape of a small part of the enzyme made DHPS resistant to sulfa drugs, it did not disrupt the enzyme's ability to combine DHPP and pABA.
"This subtle change is just enough to let DHPS prevent the antibiotic from binding to it, but not enough to disrupt the enzyme's normal work," White said. "So the bacteria evade the antibiotic and continue to make folate and survive."
Based on the new understanding of the structure of DHPS, the St. Jude team also made a new drug-like molecule that binds to a part of the enzyme that is not likely to mutate and make the bacteria resistant to antibiotic molecules designed to bind there. This molecule (5-nitro-6-methylamino-isocytosine) binds to a part of DHPS deeper within the loops and folds of the protein structure of the enzyme than the sulfa-drug binding site.
"If the drug binds to this part of DHPS, the enzyme would lose the ability to bind to DHPP and help the cell make folate," White said, "and if the enzyme mutated enough to avoid the antibiotic, the change in shape of this critical part of DHPS would destroy its ability to bind DHPP anyway. Either way, our new molecule looks like it could be the basis of a very effective new antibiotic against anthrax bacteria."
In addition, because many other infectious bacteria use DHPS to make folate, the St. Jude study holds promise for solving the growing problem of antibiotic resistance among microorganisms causing tuberculosis, pneumonia and a variety of other diseases, according to Kerim Babaoglu, the paper's first author.
"The pharmaceutical industry is reducing its investment in developing new antibiotics," Babaoglu said. "So it's important that academic institutions fill in the research gap to ensure we will have effective treatments for these infections in the years ahead."
Babaoglu is a graduate student in the interdisciplinary graduate program jointly administered by St. Jude and the University of Tennessee in Memphis.
Other authors of the paper are Jianjun Qi and Richard E. Lee. This work was supported in part by NIAID, an NIH Cancer Center (CORE) Support Grant and ALSAC.
St. Jude Children's Research Hospital
St. Jude Children's Research Hospital is internationally recognized for its pioneering work in finding cures and saving children with cancer and other catastrophic diseases. Founded by late entertainer Danny Thomas and based in Memphis, Tennessee, St. Jude freely shares its discoveries with scientific and medical communities around the world. No family ever pays for treatments not covered by insurance, and families without insurance are never asked to pay. St. Jude is financially supported by ALSAC, its fundraising organization. For more information, please visit http://www.stjude.org.
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