Cloning and expression of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii: evidence of the divergence of enzymes in the class of two-protein component aromatic hydroxylases.

The genes encoding for the reductase and oxygenase components of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii were cloned and expressed in an E. coli system. The recombinant enzymes were purified and shown to have the same catalytic properties as the native enzyme. Sequence analysis and biochemical studies indicate that the enzyme represents a novel prototype of enzyme in the two-protein component class of aromatic hydroxylases. The C2 component shows little similarity to other oxygenases in the same class, correlating with its uniquely broad flavin specificity. Analysis of the C1 reductase sequence indicates that the binding sites of flavin and NADH mainly reside in the N-terminal half while the C-terminal half may be responsible for HPA-stimulation of NADH oxidation.