By David N. Leff
Instead of expecting little green men with antennae sprouting from their heads, scanners of outer space for life as we know it might do better to look out for one atom of carbon hitched to three of hydrogen, -CH3.
Kids in chemistry class learn to recognize this cluster of four atoms as a methyl group. Bioscientists know it as the star player of methylation, a universal life process practiced all the way from viruses to plants to mammals.
Structural biologist P. Lynne Howell knows methylation as "taking a -CH3 methyl group from the S-adenosylmethionine and adding it to a biological molecule of some sort. It could be DNA, RNA, small molecules, polysaccharides; the list is endless. It ends up as S-adenosylhomocysteine."
Howell is a member of the Research Institute at the Hospital for Sick Children, in Toronto.
S-adenosylhomocysteine (SAM for short) is a complex of the nucleoside adenosine with homocysteine, which in turn is a form of the amino acid cysteine. "High levels of homocysteine," Howell observed, "have been very much in the news as an independent risk factor for cardiovascular disease. Most people can reduce homocystein levels by consuming vitamin B6, B12 or folic acid.
"But some individuals," she went on, "suffer from a genetic disorder of one enzyme in the methylation pathway, so they don't respond to vitamin therapy." Howell and her collaborators are researching an adjacent catalyst in the pathway.
"Some of the inhibitors that have already been designed to our SAM enzyme," she told BioWorld Today, "are effective at lowering homocysteine levels — certainly in a mouse model."
Howell is senior author of an article in the May 1998 issue of Nature Structural Biology. Its title: "Structure determination of selenomethionyl S-adenosylhomocys-teine hydrolase using data at a single wavelength."
As for the adenosine portion of SAM, when its metabolism goes awry, the result is severe combined immunodeficiency, also known as SCID — caused, Howell observed, "by a genetic defect in the enzyme adenosine deaminase. It happens to be the only protein we have that is regularly used in human therapy, to treat the deficiency.
"High levels of adenosine," she explained, "unless metabolized, will alter the equilibrium between the products of our SAM hydrolase enzyme and its substrate, thus inactivating the methylation."
But Howell and her co-authors are out for bigger game — in terms of human populations affected — in Africa.
Add Ebola Virus To List Of Targets
"DNA from a parasite," she noted, "requires methylation before it can replicate."
Three African parasites in particular are targeted by her group's rational drug design program: Plasmodium, which causes malaria; Trypanosomes, vectors of African sleeping sickness, and Leishmania, perpetrators of leishmaniasis.
"Interestingly enough," Howell observed, "apparently some of the inhibitors designed against this enzyme are actually effective — in a mouse model — against the Ebola virus."
She pointed out that "the parasites that we're targeting all have their own hydrolases. If we can design drugs that will inhibit the parasitic hydrolase more specifically than the human enzyme, then maybe we'll end up with a therapeutic preparation of use against a number of these parasites."
Most of their drug-design efforts, she observed, "are obviously targeted toward the Third World. They're not diseases that are common or prevalent in industrial countries."
Her group is funded by the U.S. National Institutes of Health. "I think the reason some of the pharmaceutical companies are not interested," Howell suggested, "is because there is no market for the drugs that we could design, even though they could have great potential therapeutic value for a large percentage of the world's population.
"We're funded because there are a number of other aspects of how you control methylation. There's a Japanese company," she vouchsafed, "with which my co-authors and I collaborate. They are interested in the homocysteine angle."
Having solved the SAM-hydrolase molecular structure by X-ray crystallography, Howell recounted, "basically what we've got now, for the first time, is the picture of what that enzyme looks like. Now we can propose its mechanism of action, and using that information about its active site, hopefully build a better inhibitor.
"Researchers," she went on, "have been working on this, designing drugs and inhibitors, probably for the last 15 or 20 years. But for that long period of time nobody's been able to crystallize this enzyme successfully. We managed to come up with a protocol that enabled us to get good quality crystals, which led ultimately to structure-function determination.
"The way we determined the structure is very neat," she said. "We got the crystal in January 1997, and determined its three-dimensional structure in July."
Heavy Metal Innovations In Structure Analysis
"First," Howell recounted, "we had to crystallize the protein. Second, collect data, giving us amplitudes of diffractive X-rays. An X-ray is a wave function," she explained.
"What we got was just the amplitude of the wave. Nothing about its phase. A number of people in the field have been trying to work out routine methods of solving the phase problem. What they have done is take a heavy metal, like platinum or gold, soak it into the crystal, and collect the data in both the presence and absence of this heavy metal. And the differences give an initial phase for the amplitude.
"The idea of soaking in this heavy atom," she said, "is very hit-and-miss. We actually spent almost two years trying to do it this way. Even after we'd got our crystals, it was not straightforward.
"As we reported in the journal, instead of relying on the somewhat random substitution of heavy metals into a crystal, we systematically replaced our initial methionines with selenomethionines. Then we grew up a recombinant protein, and supplemented the media with selenomethionine at 30 positions, an unprecedentedly large number of seleniums.
"And this will have considerable significance," Howell pointed out, "especially as the Human Genome Project comes to completion, and we finally have the whole human genome sequenced. So when people start to know what these proteins do, one of the things they're going to turn to is structural studies. So here they have a method of rapidly being able to determine structure.
"This could be of great potential benefit to people interested in the structure-function relationships of their pet protein," she concluded. *