2-Oxo-hept-4-ene-1,7-dioate hydratase from Escherichia coli C converts 2-oxo-hept-4-ene-1,7-dioate to 2-oxo-4-hydroxy-hepta-1,7-dioate by the addition of water using magnesium as a cofactor. The enzyme is one of a set of inducible enzymes, known collectively as the homoprotocatechuate meta-fission pathway. The entire pathway enables the organism to utilize aromatic amino acids as its sole sources of carbon and energy. Expression and purification of 2-oxo-hept-4-ene-1,7-dioate hydratase to homogeneity permitted kinetic, isotopic labeling, and stereochemical studies. Kinetic studies show that the enzyme processes either 2-oxo-hept-4-ene-1,7-dioate or 2-hydroxy-2,4-heptadiene-1,7-dioate to product with comparable facility. Isotope labeling studies show that the hydratase catalyzes the incorporation of a solvent deuteron at both C-3 and C-5 when the reaction is performed in (H2O)-H-2. The enzyme also accelerates the exchange of the C-3 proton of the alternate substrate 2-oxo-1,7-heptadioate with solvent deuterons. The results are consistent with a mechanism in which the enzyme catalyzes the isomerization of 2-oxo-hept-4-ene-1,7-dioate to its cr,P-unsaturated ketone followed by the Michael addition of water. Whether this mechanistic sequence involves a one-base or a two-base mechanism is not yet known.