Phenol hydroxylase from Acinetobacter radioresistens is a multicomponent enzyme - Purification and characterization of the reductase moiety

This paper reports the isolation and characterization of phenol hydroxylase (PH) from a strain belonging to the Acinetobacter genus. An Acinetobacter radioresistens culture, grown on phenol as the only carbon and energy sourc e, produced a multicomponent enzyme system, located in the cytoplasm and in ducible by the substrate, that is responsible for phenol conversion into ca techol. Because of the wide diffusion of phenol as a contaminant, the prese nt work represents an initial step towards the biotechnological treatment o f waste waters containing phenol. The reductase component of this PH system has been purified and isolated in large amounts as a single electrophoreti c band. The protein contains a flavin cofactor (FAD) and an iron-sulfur clu ster of the type [2Fe-2S]. The function of this reductase is to transfer re ducing equivalents from NAD(P)H to the oxygenase component. In vitro, the e lectron accepters can be cytochrome c as well as other molecules such as 2, 6-dichlorophenolindophenol, potassium ferricyanide, and Nitro Blue tetrazol ium. The molecular mass of the reductase was determined to be 41 kDa by SDS /PAGE and 38.8 kDa by gel permeation; its isoelectric point is 5.8. The N-t erminal sequence is similar to those of the reductases from A. calcoaceticu s NCIB 8250 (10/12 identity) and Pseudomonas CF600 (8/12 identity) PHs, but much less similar (2/12 identity) to that of benzoate dioxygenase reductas e from A. calcoaceticus BD413, Similarly, the internal peptide sequence of the A. radioresistens PH reductase displays a good level of identity (9/10) with both A. calcoaceticus NCIB 8250 and Pseudomonas CF600 PH reductase in ternal peptide sequences but a poorer similarity (3/10) to the internal pep tide sequence of benzoate dioxygenase reductase from A. calcoaceticus BD413 .