Ying Lou, a postdoctoral research fellow working in Shanklin's lab, used several independent methods of bi-molecular complementation-methods that produce a signal if two test proteins come together-to establish which desaturases interact with themselves or others.
The scientists found that all the plant membrane desaturases they examined are capable of forming self-associating dimers in plant cells-pairings of two identical desaturase enzyme molecules. They also found that certain desaturases with different functions could also pair up, but others could not.
"The naturally pairing enzymes turn out to have interesting patterns," Shanklin said. "They are found in the same subcellular locations within the cell, and are involved in subsequent steps of the same metabolic pathway, suggesting a physiological driver for the observed pairings.
"Other pairings between very similar desaturases from different locations that we expected to pair up didn't," he added.
To test the idea that the paired enzymes were working together, the scientists conducted another series of experiments called metabolic flux analysis, drawing on Brookhaven biochemist Jorg Schwender's expertise. This method follows mass-labeled compounds through the various reaction pathways.
"Think of a city map with lots of ways to get from A to B. This method traces how many molecules travel along each route," Schwender said.
The analysis showed that one of the natural enzyme pairings performed two steps of a particular metabolic process without releasing an intermediate product.
"This was clear evidence that these two linked enzymes were working in concert to channel metabolites through this metabolic pathway in an efficient manner in living plant cells," Shanklin said. "Our findings suggest genetic techniques may be used to engineer these kinds of interactions into other desaturase enzymes-in
|Contact: Karen McNulty Walsh|
DOE/Brookhaven National Laboratory