Purification and properties of hydroquinone hydroxylase, a FAD-dependent monooxygenase involved in the catabolism of 4-hydroxybenzoate in Candida parapsilosis CBS604

The ascomycetous yeast Candida parapsilosis CBS604 catabolizes 4-hydroxyben zoate through the initial formation of hydroquinone (1,4-dihydroxybenzene). High levels of hydroquinone hydroxylase activity are induced when the yeas t is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, 1,3-dihydrox ybenzene or 1,4-dihydroxybenzene as the sole carbon source. The monooxygena se constitutes up to 5% of the total amount of protein and is purified to a pparent homogeneity in three chromatographic steps. Hydroquinone hydroxylas e from C. parapsilosis is a homodimer of about 150 kDa with each 76-kDa sub unit containing a tightly noncovalently bound FAD. The flavin prosthetic gr oup is quantitatively resolved from the protein at neutral pH in the presen ce of chaotropic salts. The apoenzyme is dimeric and readily reconstituted with FAD. Hydroquinone hydroxylase from C. parapsilosis catalyzes the ortho-hydroxyla tion of a wide range of monocyclic phenols with the stoichiometric consumpt ion of NADPH and oxygen. With most aromatic substrates, no uncoupling of hy droxylation occurs. Hydroxylation of monofluorinated phenols is highly regi ospecific with a preference for C6 hydroxylation. Binding of phenol highly stimulates the rate of flavin reduction by NADPH. At pH 7.6, 25 degreesC, t his step does not limit the rate of overall catalysis. During purification, hydroquinone hydroxylase is susceptible towards limite d proteolysis. Proteolytic cleavage does not influence the enzyme dimeric n ature but results in relatively stable protein fragments of 55, 43, 35 and 22 kDa. N-Terminal peptide sequence analysis revealed the presence of two n ick sites and showed that hydroquinone hydroxylase from C. parapsilosis is structurally related to phenol hydroxylase from Trichosporon cutaneum. The implications of these findings for the catalytic mechanism of hydroquinone hydroxylase are discussed.