The Purification and Properties of 2,4‐Dichlorophenol Hydroxylase from a Strain of Acinetobacter Species

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Abstract
2,4-Dichlorophenol hydroxylase was purified 13-fold from Acinetobacter grown on 2,4-D as sole C source. The enzyme was estimated to be 80-90% pure by electrophoresis. The enzyme has a relative MW of .apprx. 240,000 and consists of 4 subunits of identical size. The enzyme contains FAD as the prosthetic group. FAD could not be replaced by riboflavin or FMN in reconstituting active enzyme from apoenzyme. The reaction catalyzed is an NADPH-dependent hydroxylation of 2,4-dichlorophenol with the formation of 3,5-dichlorocatechol as product. The reaction stoichiometry is typical of a monooxygenase with an external electron donor. NADPH is the preferred reduced pyridine nucleotide substrate but the enzyme can function with NADH. The enzyme possesses broad effector specificity. In addition to 2,4-dichlorophenol, 4-chlorophenol and 4-chloro-2-methylphenol are true substrates for the enzyme. A number of non-substrate effectors was found. The enzyme is sensitive to thiol-inhibiting reagents.