Molecular insights into fluorine chemistry in living systems

Based on its unique elemental properties, fluorine has emerged as an important design element in synthetic pharmaceuticals, but also a difficult substituent to incorporate into small molecules using synthetic chemistry. Synthetic biology approaches represent an attractive alternative that could allow us to harness the catalytic power and specificity of enzymes for fluorine incorporation into complex small molecules. However, fluorinated natural products are rare and derive from a single pathway for biosynthesis of fluoroacetate, an antimetabolite poison. The fluoroacetate-producing bacterium Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that is involved in fluoroacetate resistance. Using FlK as a model system, we have focused on understanding enzymatic fluorine selectivity with the goal of defining principles for the design of fluorine-specific enzymes that can be applied in the development of new methods for preparation of fluorinated molecules./nWe have demonstrated that FlK exhibits a remarkable 106-fold selectivity for hydrolysis of fluoroacetyl-CoA compared to acetyl-CoA, an abundant central metabolite and cellular competitor. This selectivity is based on a decrease in KM (102) and an increase in kcat (104) for the fluorinated substrate. Through x-ray crystallographic studies, we have identified a unique hydrophobic `lid' in our crystal structure of FlK that shields the active site from water. Mutation of a key lid residue (Phe 36) led to a loss of fluorine-based binding specificity, which was correlated with the appearance of additional ordered water molecules at the active site in the mutant crystal structure. A crystal structure of the FlK product complex revealed that Phe 36 is dynamic, undergoing a conformational change when the active site is occupied with product. By studying the binding of a series of non-hydrolyzable substrate analogs, we have shown that the lid confers the fluorinated substrate with an entropic binding advantage that is lost in lid mutants, which may be related to water release from the `polar hydrophobic' C-F unit. Kinetic studies revealed that Phe 36 controls the substrate off rate, providing the fluorinated substrate with a kinetic as well as a thermodynamic advantage./nWe have also shown that catalytic specificity in FlK is based on utilization of an unusual reaction pathway initiated by deprotonation at the α-carbon for hydrolysis of fluoroacetyl-CoA but not acetyl-CoA. The enolate that is formed can then breakdown through a putative ketene intermediate to give the same product that would be produced by a canonical hydrolysis mechanism. FlK therefore represents a rare example in which the existence of two reaction pathways in the same active site controls substrate selectivity rather than product outcome. Although catalytic fluorine discrimination occurs mainly in one step of the reaction mechanism, we have demonstrated that fluorine is specifically recognized by the enzyme in both the acylation and deacylation steps of the reaction mechanism. These studies have defined the basis of fluorine selectivity in a naturally occurring enzyme-substrate pair, with implications for drug design and development of fluorine-selective biocatalysts.

Advisor: Chang, Michelle CY