Isolation and partial characterization of an extradiol non-haem iron dioxygenase which preferentially cleaves 3-methylcatechol.
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.
Wallis
M G
MG
Department of Chemistry, University of Edinburgh, U.K.
Chapman
S K
SK
D000595:Amino Acid Sequence; D050557:Catechol 2,3-Dioxygenase; D002396:Catechols / Q000378:metabolism*; D049308:Dioxygenases; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010105:Oxygenases / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins; D013379:Substrate Specificity
D002396:Catechols; D011994:Recombinant Proteins; C031136:3-methylcatechol; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase