The 4-Oxalocrotonate Tautomerase- and YwhB-Catalyzed Hydration of 3E-Haloacrylates:  Implications for the Evolution of New Enzymatic Activities

Abstract

4-Oxalocrotonate tautomerase (4-OT) catalyzes the conversion of 2-oxo-4E-hexenedioate to 2-oxo-3E-hexenedioate through the intermediate, 2-hydroxy-2,4E-hexadienedioate. 4-OT and a homologue found in Bacillus subtilis (designated YwhB) share sequence identity and two key catalytic groups, Pro-1 and Arg-11, with the two subunits comprising trans-3-chloroacrylic acid dehalogenase (CaaD). 4-OT and YwhB have now been found to display a low-level hydratase activity, resulting in the dehalogenation of 3E-haloacrylates. The enzymes are highly selective for the (E)-isomer, and Pro-1 is critical for the activity while an arginine is likely required. Two mechanisms are proposed in which Pro-1 functions as a general base or a general acid catalyst and, along with the arginine, facilitates the Michael addition of water. Both mechanisms suggest an intriguing route for the evolution of the CaaD activity. One or more mutations could decrease the hydrophobic environment of the active site, which would make it more favorable for a hydrolytic reaction, thereby raising the pKa of Pro-1 and increasing the concentration of enzyme in the reactive form.

Phenylpyruvate Tautomerase Activity of trans-3-Chloroacrylic Acid Dehalogenase: Evidence for an Enol Intermediate in the Dehalogenase Reaction?

“…These observations established a functional link between the tautomerases and the dehalogenases (47). The observation of a PPT activity for CaaD now establishes a functional link between CaaD and the tautomerases.…”

Divergence and Convergence in Enzyme Evolution: Parallel Evolution of Paraoxonases from Quorum-quenching Lactonases

“…Nonetheless, the functional, structural, and laboratory evolutionary linkages between these two seemingly unrelated classes of enzymes, lactonases and paraoxonases, suggest that the parallel evolution of paraoxonases in two different superfamilies is more than a coincidence. It therefore appears that the identification of patterns of overlapping activities, both native and promiscuous, between different families within the same superfamily constitutes a powerful means of deciphering evolutionary relationships (12,68,(73)(74)(75)(76). As discussed here, these patterns relate to specific overlaps between transition states and/or intermediates of different reactions (see also Refs.…”

Kinetic and Structural Characterization of a Heterohexamer 4-Oxalocrotonate Tautomerase from Chloroflexus aurantiacus J-10-fl: Implications for Functional and Structural Diversity in the Tautomerase Superfamily,

“…Hence, it was proposed that Arg-11 or Arg-39 interacted with the C-1 carboxylate group of the substrate, thereby polarizing the α,β-unsaturated acid and creating a partial positive charge at C-3 (Scheme 5). Water could then attack at C-3 with (or without) the assistance of Pro-1 (22). Further support for this mechanism came from the observation that changing Leu-8 in 4-OT to an arginine enhanced the low level CaaD activity (14).…”

“…Malonate semialdehyde is not sufficiently stable to accumulate in quantities detectable by 1 H NMR spectroscopy in the course of the lengthy incubation periods (22). …”