How enzymes break carbon-fluorine bonds

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Dehalogenase enzymes break carbon-halogen bonds. Most remarkable are enzymes able to break the most stable bond in organic chemistry, the carbon-fluorine bond. Supported by an NSERC Strategic Grant and in collaboration with E. Edwards (U of T) and A.Yakunin (SGC), we screened microbial genomes for dehalogenases. We raised the number of confirmed dehalogenases from 2 to 20, 9 of them defluorinases and 4 representing the first defluorinase members of the L-2-haloacid dehalogenase protein family. We will determine the molecular interactions that form the basis of their catalytic power, contributing to biodegradation solutions for most recalcitrant environmental pollutants. We have used ITC to determine kinetic parameters of native and mutant enzymes. The fluoroacetate dehalogenase RPA1163 from R. palustris was characterized in detail. Its static high-resolution crystal structures (~ 1.2 Å; native and mutants) with various ligands provided a set of 'snapshots' of the catalytic intermediates. They also indicated transient binding sites as the substrate approaches the catalytic machinery and rotational movements upon transformation. Our static analyses showed that the crystals accommodate the complete catalytic cycle without compromising diffraction power. We have synthesized the "caged" 1-(2-nitrophenyl) ethyl-derivative of the substrate. Short UV-laser pulses can transform this "caged compound" into the substrate, initiating its binding and catalytic conversion. Applying time- resolved (TR)-Laue techniques, we will collect diffraction data as a function of time since initiation and convert them to a "movie" of the catalytic reaction, allowing observation in almost atomic detail. We collaborate with the world's foremost experts in laser-triggered TR-Laue-diffraction to accelerate our progress and secure timely access to their superb resources. Our system will also serve as a test case for developing monochromatic methods for TR crystallography. In addition, we plan to engineer a light-sensitive dehalogenase by fusing a light-trigger LUV domain to the defluorinase, which will allow us to preform the substrate complex, thereby shifting the reaction from a bimolecular to a unimolecular one, allowing for easier data interpretation.