Purification and characterization of 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase.
The Alcaligenes eutrophus 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase, encoded by the tfdA gene of plasmid pJP4, is an Fe(II)-dependent enzyme that catalyzes the conversion of 2,4-dichlorophenoxyacetate to 2,4-dichlorophenol and glyoxylate concomitant with the decomposition of alpha-ketoglutarate to form succinate and carbon dioxide (Fukumori, F., and Hausinger, R. P. (1993) J. Bacteriol. 175, 2083-2086). Using recombinant Escherichia coli cells that overexpress the tfdA gene, the thermolabile enzyme (stable only up to 30 degrees C) was purified to apparent homogeneity (specific activity of 16.9 mumol of substrate converted min-1 mg of protein-1) by a simple two-step procedure. The native protein has an apparent M(r) of 50,000 +/- 2,500, consistent with a homodimeric structure. Ferrous ion is absolutely required for activity and cannot be replaced by several other divalent cations tested. Ascorbic acid stimulates dioxygenase activity and reduces the rate of enzyme inactivation by a metal ion-mediated process. The enzyme exhibits maximum activity at pH 6.5-7, however, it is stable over a pH range of 6.5-11. Although capable of hydroxylating a wide range of phenoxyacetates and related compounds, the enzyme exhibits the greatest affinity (Km 17.5 +/- 1.0 microM) and highest catalytic efficiency for 2,4-dichlorophenoxyacetate. Similarly, alpha-ketoglutarate is the preferred co-substrate (Km 3.20 +/- 0.54 microM) for the enzyme, but it can utilize a range of other alpha-ketoacids with lower efficiency. Results from chemical modification studies are consistent with the presence of multiple essential histidine residues in the enzyme.
Fukumori
F
F
Center for Microbial Ecology, Michigan State University, East Lansing 48824.
Hausinger
R P
RP
D000421:Alcaligenes / Q000201:enzymology*; D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D006899:Mixed Function Oxygenases; C084087:2,4-dichlorophenoxyacetate-alpha-ketoglutarate dioxygenase