Andre's work pointed to a fatty acid that occurs fairly late in the production process, coupled to a carrier protein (essential to making the oily substance soluble in water), as the key intermediate that puts the brakes on oil production. It's the first "desaturated" fatty acid - the first one with a double bond between two of the carbon atoms, formed after all 18 carbon atoms are added to the chain.
Then, knowing that this intermediate somehow sent the "slow-down" signal, the team sought to determine its "target" - how it actually inhibits oil synthesis. They knew from other biochemical feedback loops that the likely target would be an enzyme early in the synthesis pathway. But they wanted to figure out exactly which one.
To do this, they monitored the production process by labeling the intermediates one at a time with a radioactive form of carbon while also feeding the cells an excess of the "slow-down" signaling fatty acid. If the label from the intermediate ended up in the oil product, the "slow-down" signal had to have its effect prior to that step.
The first two experiments gave them the answer: When they labeled the first compound in the synthesis pathway, which is acetate, very little labeled carbon ended up in the oil and oil production was strongly reduced. But if they fed the second compound, labeled malonate, the labeled carbon quickly entered the oil.
"From these findings we concluded that the accumulation of the first desaturated fatty acid in the synthesis process inhibits the enzyme that operates at the first step, which converts acetate to malonate," Shanklin said. "That enzyme is Acetyl-CoA carboxylase, or ACCase."
The next step was to make extracts of the tissue culture and directly measure A
|Contact: Karen McNulty Walsh|
DOE/Brookhaven National Laboratory