Stereochemical and isotopic labeling studies of 4-oxalocrotonate decarboxylase (EC 4.1.1.-; 4-OD) and vinylpyruvate hydratase (EC 4.2.1.-; VPH) from Pseudomonas putida mt-2 have been completed. The two enzymes, reportedly a complex, catalyze successive reactions in the catechol meta-fission pathway and convert 2-oxo-3-hexenedioate (1) to 2-oxo-4-hydroxypentanoate (2) using either manganese or magnesium as a cofactor. 2-Oxo-4-pentenoate (3) and 2-hydroxy-2,4-pentadienoate (4) have been detected by UV and H-1 NMR spectroscopy in the 4-OD-catalyzed decarboxylation of 1. Incubation of 4 with 4-OD in (H2O)-H-2 resulted in its highly stereoselective ketonization to afford (3S)-[3-H-2]3. A reasonable hypothesis to explain these observations is that 4-OD catalyzes the decarboxylation of 1 to 3 through the intermediacy of the dienol 4. It was further shown that 4-OD converts (5S)-[5-H-2]1 to 4E-[5-H-2]4 in (H2O)-H-2. These stereochemical results coupled with the previously established S configuration of [5-H-2]1 indicate that the loss of carbon dioxide and the incorporation of a deuteron occur on the same side of the dienol intermediate. The product of the 4-OD/VPH complex was also isolated and identified unequivocally as (4S)-2. Finally, it was determined by an O-18 labeling experiment that the hydroxyl group at C-4 of 2 is derived from solvent water and that it is unlikely that either 4-OD or VPH utilizes a Schiff base intermediate.