Engineered bi-functional enzyme increases output of bio-alkanes; “protection via inhibitor metabolism”
8 February 2013
Researchers at Brookhaven National Laboratory studying an enzymatic pathway that naturally produces alkanes—long carbon-chain molecules that could be a direct replacement for the hydrocarbons in gasoline—have discovered why the natural reaction typically stops after three to five cycles, and have devised a strategy to keep the reaction going. The findings, published in a paper in the Proceedings of the National Academies (PNAS), could bolster work in using bacteria, algae, or plants to produce biofuels that need no further processing.
The cyanobacterial pathway, consisting of acyl–Acyl Carrier Protein reductase and an aldehyde-deformylating oxygenase (ADO), converts acyl–Acyl Carrier Proteins into corresponding n-1 alkanes via aldehyde intermediates in an oxygen-dependent manner. In vitro, ADO turns over only three times; however, the addition of more ADO to exhausted assays results in additional product formation. ADO’s resemblance to a group of enzymes with which the Brookhaven scientists were familiar drew them into working to discover 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.—Dr. John Shanklin, who led the research
They determined that one of the electron transport proteins was interacting with oxygen to produce hydrogen peroxide (H2O2), and the buildup of hydrogen peroxide was poisoning the ADO enzyme, completely inhibiting its activity.
To confirm that hydrogen peroxide buildup was the problem and to simultaneously test whether its depletion might enhance alkane production, Shanklin and his team added another enzyme, catalase (CAT), which metabolizes hydrogen peroxide to oxygen and water. With both enzymes, the reaction ran for more than 225 cycles, rather than stopping after three.
The scientists then made a bi-functional enzyme by linking the two together, reasoning 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 reaction going.
Living cells often contain levels of hydrogen peroxide sufficient to cause ADO inhibition. So there was a question about whether the dual enzyme would increase alkane production under these natural conditions.
Results to date have been encouraging: In experiments in test tubes and pilot studies in bacteria, the bi-functional enzyme resulted in at least a five-fold increase in alkane production compared with ADO alone. And, 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.
Now the scientists are working to install the combo enzyme in algae or green plants.
While ADO-containing bacteria convert sugar that we feed to them into alkanes, it would be much more efficient to produce alkanes in photosynthetic organisms using carbon dioxide and sunlight.—John Shanklin
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.
The research was funded by the DOE Office of Science.
Carl Andre, Sung Won Kim, Xiao-Hong Yu, and John Shanklin (2013) Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H2O2 to the cosubstrate O2. PNAS doi: 10.1073/pnas.1218769110
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