The scientists identified one position, far from the active site, where the computer model indicated that switching a single amino acid would change the orientation of the bound fatty acid with respect to the active site. Could this distant amino-acid location remotely control the site of double bond placement?
To test this hypothesis, the scientists engineered a new desaturase, swapping out the aspartic acid normally found at that location in the delta-9 castor desaturase for the lysine found in the delta-4 ivy desaturase. The result: an enzyme that was castor-like in every way, except that it now seemed able to desaturate the fatty acid at the delta-4 carbon location. "It's quite remarkable to see that changing just one amino acid could have such a striking effect," Shanklin said.
The computational modeling helped explain why: It showed that the negatively charged aspartic acid in the castor desaturase ordinarily repels a negatively charged region on the carrier protein, which leads to a binding orientation that favors delta-9 desaturation; substitution with positively charged lysine results in attraction between the desaturase and carrier protein, leading to an orientation that favors delta-4 desaturation.
Understanding this mechanism led Ed Whittle, a research associate in Shanklin's lab, to add a second positive charge to the castor desaturase in an attempt to further strengthen the attraction. The result was a nearly complete switch in the castor enzyme from delta-9 to delta-4 desaturation, adding compelling support for the remote control hypothesis.
"I really admire Ed's persistence and insight in taking what was already a striking result and pushing it even further to completely change the way this enzyme functions," Shanklin said.
"It's very rewarding to have finally solved this mystery, which would not have been possible without
|Contact: Karen McNulty Walsh|
DOE/Brookhaven National Laboratory