Stereospecific Ketonization of 2-Hydroxymuconate by 4-Oxalocrotonate Tautomerase and 5-(Carboxymethyl)-2-hydroxymuconate Isomerase

4-Oxalocrotonate tautomerase from Pseudomonas putida mt-2 and 5-(carboxymethyl)-2-hydroxymuconate isomerase from Escherichia coli C catalyze the same reaction on substrates that differ only by a carboxymethyl group. While the structural resemblances between the substrates for these two proteins suggest that the enzymes might be evolutionarily related, the existing literature does not indicate an obvious link. It was found that both enzymes ketonize 2-hydroxymuconate (1)-the substrate for 4-oxalocrotonate tautomerase. In an effort to uncover similarities in the mechanisms, the stereochemical courses of both enzymatic reactions were examined, utilizing 1 in (H2O)-H-2. In each case, the product, 2-oxo-3-(E)-hexenedioate (2), was trapped and processed to [2-H-2(1)]glutaric acid by chemical degradative procedures. The configuration was established by comparing the molar ellipticity of the isolated glutaric acid to that of a sample generated by a stereoselective synthesis. It is concluded that 4-OT and CHMI ketonize 1 stereospecifically to (S)-2-oxo-3-(E)-[5-H-2(1)]hexenedioate. The mechanism and the evolution of 4-OT and CHMI and their respective pathways are discussed in the context of these results.