How enzymes break carbon-fluorine bonds

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Canada has identified and classified over 22,000 contaminated or suspected contaminated sites. Many of them harbour halogenated hydrocarbons, contaminants that are highly resistant to natural breakdown as well as remediation efforts. Some of these compounds can even be found in the blood of polar bears. Dehalogenases are enzymes that break carbon-halogen bonds; they are being tested for restoring such contaminated sites. With our work, we intend to improve the use of these proteins as more environmentally friendly means of remediation. ******Many bacteria produce such dehalogenases but it is difficult to identify them simply by sequencing the genome because they look very similar to other proteins that are not able to perform the same catalysis. Of special interest are enzymes that can break the strongest bond in organic chemistry, the one between carbon and fluorine. To provide initial insight towards the future development of biodegradation solutions for some of the most recalcitrant environmental pollutants, our lab, in collaboration with our colleague E. Edwards, screened bacteria for dehalogenase activity. We increased the number of confirmed dehalogenases from 2 to 20, 9 of them defluorinases. Four of the latter belong to a protein family called HAD and are its first members identified as defluorinases. Our research aims to determine why these enzymes are such potent catalysts. ******A dehalogenase belonging to another protein family called ABH was isolated from the marsh bacterium Rhodopseudomonas palustris and characterized in atomic detail by X-ray crystallography. The enzyme converts fluoroacetate, a toxic compound found in grasses in Australia, Brazil, and Africa and used as a pesticide, to the harmless compound glycolate. We were able to visualize how the protein interacts with its substrate to assemble a set of `snapshots' of the catalytic reaction. We could also show that one can run the complete catalytic cycle in the crystals without breaking them. We have synthesized modified substrates that can be turned back into active substrates by exposing them to short pulses of laser light. When this is done in crystals and X-rays are shone on these one can watch the `molecular movie' of the ongoing catalytic reaction, allowing the observation of the chemical transformation in almost atomic detail. ******By constructing chimeras of bacterial or plant proteins that change their shape upon illumination with various dehalogenases, we will test their use as light-activated/switchable constructs that could add a reversible component to the dehalogenase reaction. We will also test crystals of dechlorinating enzymes incapable of defluorination for their ability to accommodate the full catalytic cycle in the crystal lattice. By comparing both resulting "molecular movies", we hope to pinpoint the structural features that make a defluorinase a defluorinase.**