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Scientists turn toxic by-product into biofuel booster
Date:2/4/2013

DO's remarkable resemblance to a group of enzymes the Brookhaven scientists were familiar with drew them deeper into the mystery of why the enzyme stopped working.

"We set to work to try to understand the biochemistry of ADO because it is so similar to the desaturase enzymes that we study, but performs a very different and interesting reaction," Shanklin said.

The key discovery -- that the alkane-producing system creates a by-product that's toxic to the ADO enzyme -- was unexpected. It was also the key to solving the turnover problem.

To simplify the analysis of ADO, the scientists tested whether they could substitute hydrogen peroxide for the electron transfer proteins and oxygen normally required for the alkane-producing reaction-an approach that had worked for a related enzyme. But instead of stimulating alkane production, no alkane at all was produced, and in control experiments containing all the components plus hydrogen peroxide, alkane production was also blocked.

"It turns out one of the electron transport proteins was interacting with oxygen to produce hydrogen peroxide, and the buildup of hydrogen peroxide was 'poisoning' the ADO enzyme, completely inhibiting its activity," Shanklin said.

To confirm that hydrogen peroxide buildup was the problem and to simultaneously test whether its depletion might enhance alkane production, Shanklin and his team tried adding another enzyme, catalase, which metabolizes hydrogen peroxide to oxygen and water.

"When we added both enzymes, instead of the reaction turning over three times before stopping, it ran for more than 225 cycles," Shanklin said.

So the scientists decided to make a "bi-functional" enzyme by linking the two together.

"We reasoned that with the ADO and catalase enzymes linked, as the hydrogen peroxide concentration near the enzyme increases, the catalase could convert it to oxygen, mitigating the inhibition and thereby keeping the r
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Contact: Karen McNulty Walsh
kmcnulty@bnl.gov
631-344-8350
DOE/Brookhaven National Laboratory
Source:Eurekalert

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