Abstract
A purification procedure has been developed for an extradiol dioxygenase expressed in Escherichia coli, which was originally derived from a Pseudomonas putida strain able to grow on toluidine. Physical and kinetic properties of the enzyme have been investigated. The enzyme has a subunit Mr of 33,500 +/- 2000 by SDS/polyacrylamide-gel electrophoresis. Gel filtration indicates a molecular mass under non-denaturing conditions of 120,000 +/- 20,000. The N-terminal sequence (35 residues) of the enzyme has been determined and exhibits 50% identity with other extradiol dioxygenases. Fe(II) is a cofactor of the enzyme, as it is for other extradiol dioxygenases. The reactivity of this enzyme towards catechol and methyl-substituted catechols is somewhat different from that seen for other catechol 2,3-dioxygenases, with 3-methylcatechol cleaved at a higher rate than catechol or 4-methylcatechol. Km values for these substrates with this enzyme are all around 0.3 microM. The enzyme exhibits a bell-shaped pH profile with pKa values of 6.9 +/- 0.1 and 8.7 +/- 0.1. These results are compared with those found for other extradiol dioxygenases.
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