Duggleby_1986_Microbiology_132_717

The 2-hydroxy-6-oxohepta-2,4-dienoate (HOD) hydrolase encoded by the TOL plasmid pWW0 from Pseudomonas putida mt-2 (PaWl) was purified to homogeneity. It has an Mr of 65000 and is dissociated by SDS into two subunits of equal size. Alanine was the only N-terminal residue detected, and each subunit contained one cysteine thiol group. The pH optimum for activity and for enzyme stability was around 7.5, whereas the isoelectric point was 4.7. Only the products of catechol 2,3-oxygenase action on linear chain alkylcatechols and 4-chlorocatechol served as substrates. Kinetic measurements showed that the higher activity against the ketone substrates (from 3-substituted catechols) over aldehyde substrates (from 4-substituted catechols) was the result of higher Vmax values rather than lower Km values. Antisera prepared against this purified HOD hydrolase were shown by Ouchterlony double diffusion and inhibition studies to be related to the HOD hydrolase from phenol-grown P. putida strain U (NCIB 10015) in which the enzyme is chromosomally encoded.