Shanklin and his collaborators approached the problem by studying two genetically similar desaturases that act at different locations: a castor desaturase that inserts a double bond between carbon atoms 9 and 10 in the chain (a 'delta-9' desaturase); and an ivy desaturase that inserts a double bond between carbon atoms 4 and 5 (delta-4). They reasoned that any differences would be easy to spot in such extreme examples.
But early attempts to find a telltale explanation which included detailed analyses of the two enzymes' atomic-level crystal structures turned up few clues. "The crystal structures are almost identical," Shanklin said.
The next step was to look at how the two enzymes bind to their substrates fatty acid chains attached to a small carrier protein. First the scientists analyzed the crystal structure of the castor desaturase bound to the substrate. Then they used computer modeling to further explore how the carrier protein "docked" with the enzyme.
"Results of the computational docking model exactly matched that of the real crystal structure, which allows carbon atoms 9 and 10 to be positioned right at the enzyme's active site," Shanklin said.
Next the scientists modeled how the carrier protein docked with the ivy desaturase. This time it docked in a different orientation that positioned carbon atoms 4 and 5 at the desaturation active site. "So the docking model predicted a different orientation that exactly accounted for the specificity," Shanklin said.
To identify exactly what was responsible for the difference in binding, the scientists then looked at the amino acid sequence the series of 360 building blocks that makes up each enzyme. They identified amino acid locations that differ between delta-9 and delta-4 desaturases, and focused on those locations that would be able to inter
|Contact: Karen McNulty Walsh|
DOE/Brookhaven National Laboratory