Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase

Abstract

4-Oxalocrotonate tautomerase (4-OT) and trans-3-chloroacrylic acid dehalogenase (CaaD) are members of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. 4-OT, from Pseudomonas putida mt-2, catalyzes the conversion of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the dienol intermediate 2-hydroxymuconate in a catabolic pathway for aromatic hydrocarbons. CaaD, from Pseudomonas pavonaceae 170, catalyzes the hydrolytic dehalogenation of trans-3-chloroacrylate in the trans-1,3-dichloropropene degradation pathway. Both reactions may involve an arginine-stabilized enediolate intermediate, a capability that may partially account for the low-level CaaD activity of 4-OT. Two active-site residues in 4-OT, Leu-8 and Ile-52, have now been mutated to the positionally conserved and catalytic ones in CaaD, alphaArg-8, and alphaGlu-52. The L8R and L8R/I52E mutants show improved CaaD activity (50- and 32-fold increases in k(cat)/K(m), respectively) and diminished 4-OT activity (5- and 1700-fold decreases in k(cat)/K(m), respectively). The increased efficiency of L8R-4-OT for the CaaD reaction stems primarily from an 8.8-fold increase in k(cat), whereas that of the L8R/I52E mutant is due largely to a 23-fold decrease in K(m). The presence of the additional arginine residue in the active site of L8R-4-OT does not alter the pK(a) of the Pro-1 amino group from that measured for the wild type (6.5 +/- 0.1 versus 6.4 +/- 0.2). Moreover, the crystal structure of L8R-4-OT is comparable to that of the wild type. Hence, the enhanced CaaD activity of L8R-4-OT is likely due to the additional arginine residue that can participate in substrate binding and/or stabilization of the putative enediolate intermediate. The results also suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.

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Figure 1

A comparison of (A) the crystal structure of wild type 4-OT (PDB Accession 4OTB) and (B) the crystal structure of the L8R-4-OT. The figure shows one active site in each structure. Except for the replacement of Leu-8 with arginine, the two structures are comparable. Ile-52 is located above Phe-50 in the 4-OT structure and near the side chain carbon atoms of Arg-39″. The movement of Arg-11 in the L8R-4-OT structure is likely due to the fact that Arg-11 is a surface residue and is responding to a different environment.

Figure 2

The pH titration curve displaying the ¹⁵N-chemical shift of the amino group of Pro-1 of L8R-4-OT versus pH. The data from this plot were used to calculate the pK_a of the amino proton of Pro-1, which is reported in the text.