PURIFICATION AND CHARACTERIZATION OF 2,4-DICHLOROPHENOXYACETATE ALPHA-KETOGLUTARATE DIOXYGENASE

The Alcaligenes eutrophus 2,4-dichlorophenoxyacetate/alpha-ketoglutara te dioxygenase, encoded by the tfdA gene of plasmid pJP4, is an Fe(II) -dependent enzyme that catalyzes the conversion of 2,4-dichlorophenoxy acetate to 2,4-dichlorophenol and glyoxylate concomitant with the deco mposition of alpha-ketoglutarate to form succinate and carbon dioxide (Fukumori, F., and Hausinger, R. P. (1993) J. Bacteriol. 175, 2083-208 6). Using recombinant Escherichia coli cells that overexpress the tfdA gene, the thermolabile enzyme (stable only up to 30-degrees-C) was pu rified to apparent homogeneity (specific activity of 16.9 mumol of sub strate 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, consist ent with a homodimeric structure. Ferrous ion is absolutely required f or activity and cannot be replaced by several other divalent cations t ested. Ascorbic acid stimulates dioxygenase activity and reduces the r ate 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 p H range of 6.5-11. Although capable of hydroxylating a wide range of p henoxyacetates and related compounds, the enzyme exhibits the greatest affinity (K(m) 17.5 +/- 1.0 muM) and highest catalytic efficiency for 2,4-dichlorophenoxyacetate. Similarly, alpha-ketoglutarate is the pre ferred co-substrate (K(m) 3.20 +/- 0.54 muM) for the enzyme, but it ca n utilize a range of other alpha-ketoacids with lower efficiency. Resu lts from chemical modification studies are consistent with the presenc e of multiple essential histidine residues in the enzyme.