Engineered enzyme converts toxic by-product into biofuel

Scientists studying an enzyme that naturally produces alkanes, long carbon-chain molecules that could be a direct replacement for the hydrocarbons in gasoline, have figured out why the natural reaction typically stops after three to five cycles. They have devised a strategy for prolonging the reaction.
The biochemical details, worked out at the U.S. Department of Energy’s Brookhaven National Laboratory and described in the Proceedings of the National Academy of Sciences, renew interest in using the enzyme in bacteria, algae, or plants to produce biofuels that need no further processing.
“Alkanes are very similar to the carbon-chain molecules in gasoline. They represent a potential renewable alternative to replace the petrochemical component of gasoline,” said Brookhaven biochemist John Shanklin, who led the research, which was conducted in large part by former Brookhaven post-doctorate Carl Andre, now working at BASF Plant Science in North Carolina, and Xiaohong Yu of Brookhaven’s Biosciences Department. “Unlike the process of breaking down plant biomass to sugars and fermenting them to ethanol,” Shanklin said, “biologically produced alkanes could be extracted and used directly as fuel.”
Recent discovery of an enzyme known as aldehyde-deformylating oxygenase (ADO), which naturally makes alkanes from precursors in certain bacteria, stimulated interest in harnessing this enzyme’s action to make liquid biofuels. But early attempts to install ADO in laboratory-based alkane ‘factories’ gave disappointing results.
Likewise, the Brookhaven team’s experiments in test tubes using substrates, synthesized with the help of Sunny Kim in Brookhaven’s Radiotracer and Biological Imaging group, yielded the same result others had observed. The enzyme mysteriously stopped working after three to five ‘turnovers’ and alkane production would cease.
Biochemical curiosity and ADO’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.
To confirm that hydrogen peroxide build-up 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. So the scientists decided to make a ‘bi-functional’ enzyme by linking the two together.
In experiments in test tubes and pilot studies in bacteria, the bifunctional enzyme resulted in at least a five-fold increase in alkane production compared with ADO alone. In addition to removing hydrogen peroxide as an inhibitor of ADO, the combo enzyme actually helps drive the alkane-producing reaction by producing oxygen, one of the key components required for activity. “This bi-functional enzyme simultaneously decreases the concentration of the inhibitor and increases the concentration of a needed reaction component by converting an inhibitor into a substrate,” Shanklin said.
Now the scientists are working to install the combo enzyme in algae or green plants. The scientists also suggest that the general approach of strategically designing fusion enzymes to break down small molecule inhibitors could be used to improve the efficiency of a wide range of reactions. Defeating natural inhibition, a process they describe as ‘protection via inhibitor metabolism’ (PIM), would allow such bifunctional enzymes to function more efficiently than their natural counterparts.
(March 28, 2013)