On the reactions of trans-3-chloroacrylic acid dehalogenase and a cis-3-chloroacrylic acid dehalogenase homologue, Cg10062 : mechanistic and evolutionary implications

The tautomerase superfamily (TSF) provides an excellent model system to study enzyme specificity, catalysis, and divergent evolution. trans-3-Cholroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), and malonate semialdehyde decarboxylase (MSAD) are three TSF members that catalyze the final reactions in the degradation of the nematocide, 1,3-dichloropropene. All three enzymes have the TSF characteristic beta-alpha-beta fold and catalytic amino terminal proline (Pro-1). Both CaaD and cis-CaaD dehalogenate their respective isomers of 3-chloroacrylic acid yielding malonate semialdehyde. Subsequently, MSAD decarboxylates malonate semialdhyde resulting in acetaldehyde and CO2. Their catalytic and substrate specificities are exquisite considering they share three key and positionally conserved residues. As part of an effort to understand how such specificity evolved, a pre-steady-state kinetic analysis of CaaD was carried out. Alongside a similar study on cis-CaaD, a fluorescent mutant of CaaD was constructed that had minimal kinetic differences from the wild-type. The mutant was validated as an accurate fluorescent reporter of change in enzyme state that allowed for the reaction to be followed using stopped-flow methods. Stopped-flow fluorescence, rapid chemical quench data and ultraviolet spectroscopy were globally fit by computational simulation. The fit resulted in a kinetic mechanism for CaaD affording detailed information about the reaction, including measuring the rate of product release, the rate of chemistry, a previously unknown partially rate-limiting step associated with a conformational change, and the definition of binding constants for both products (MSA and Br-). In addition to the dehalogenation reaction, the reaction of the fluorescent mutant with a mechanism-based inhibitor, 3-bromopropiolate, was characterized. The values for the apparent rate of inhibition and potency were defined and estimates were determined for the values of the rate of chemistry and the release of bromide. The information gathered during these inhibition experiments was used to further refine the CaaD dehalogenation mechanism eliminating ambiguities present in the initial data set. Finally, the reactions of a cis-CaaD homologue, Cg10062 from Corynebacterium glutamicum were characterized. Cg10062 shares high sequence similarity (53%) and the same six critical active site residues as cis-CaaD, but Cg10062 has poor cis-CaaD activity. Moreover, Cg10062 dehalogenates both 3-chloroacrylic acid isomers. The reactions of Cg10062 with propiolate, 2-butynoate, and 2,3 butadienoate were investigated. Cg10062 functions as a hydratase/decarboxylase using propiolate generating malonate semialdehyde and acetaldehyde. Cg10062 catalyzes a hydration-dependent decarboxylation of propiolate as exogenously added malonate semialdehyde is not decarboxylated. With 2,3 butadienoate and 2-butynoate, Cg10062 functions as a hydratase and yields only acetoacetate. Mutations to the activating residues Glu114 and Tyr103 produced a range of results from a reduction in wild-type activity to a switch of activity. Possible intermediates for the hydration and decarboxylation products can be trapped as covalent adducts to Pro-1 when NaCNBH3 is incubated with certain combinations of substrate and mutant enzymes. Three mechanisms are presented to explain these findings along with the strengths and weaknesses of each mechanism in terms of being able to account for experimental observations.