MOLECULAR CHARACTERIZATION OF 4-HYDROXYPHENYLACETATE 3-HYDROXYLASE OFESCHERICHIA-COLI - A 2-PROTEIN COMPONENT ENZYME

The nucleotide sequences of the hpaB and hpaC genes encoding the 4-hyd roxyphenylacetate 3-hydroxylase from Escherichia coli W ATCC 11105 hav e been determined. These genes appear to be part of an operon and enco de two proteins of 58,781 and 18,679 Da, respectively, that are requir ed for hydroxylase activity. This aromatic hydroxylase is NADH depende nt and uses FAD as the redox chromophore. The largest component (HpaB) has been purified by affinity chromatography in Cibacron blue. E. col i cells that express exclusively hpaB showed only a very low hydroxyla se activity that was enhanced in the presence of extracts containing t he smallest protein HpaC. This behavior resembles that of the coupling protein of the 4-hydroxyphenylacetate 3-hydroxylase from Pseudomonas putida, and it might prevent the wasteful oxidation of NADH in the abs ence of substrate. Using a promoter-probe plasmid we have demonstrated that the hpaBC operon is expressed by a promoter inducible by 4-hydro xyphenylacetic acid. A gene, named hpaA, encoding a protein homologous to the XylS/AraC family of regulators, was identified upstream of the hydroxylase operon. The role played by HpaA in the regulation of the hpaBC operon remains to be elucidated. Since HpaB is not homologous to other aromatic hydroxylases, we suggest that the E. coli 4-hydroxyhen ylacetate 3-hydroxylase is the first member of a new family of two-com ponent aromatic hydroxylases sequenced so far.