The ascomycetous yeast Candida parapsilosis CBS604 catabolizes 4-hydroxybenzoate through the initial formation of hydroquinone (1, 4-dihydroxybenzene). High levels of hydroquinone hydroxylase activity are induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, 1,3-dihydroxybenzene or 1, 4-dihydroxybenzene as the sole carbon source. The monooxygenase constitutes up to 5% of the total amount of protein and is purified to apparent homogeneity in three chromatographic steps. Hydroquinone hydroxylase from C. parapsilosis is a homodimer of about 150 kDa with each 76-kDa subunit containing a tightly noncovalently bound FAD. The flavin prosthetic group is quantitatively resolved from the protein at neutral pH in the presence of chaotropic salts. The apoenzyme is dimeric and readily reconstituted with FAD. Hydroquinone hydroxylase from C. parapsilosis catalyzes the ortho-hydroxylation of a wide range of monocyclic phenols with the stoichiometric consumption of NADPH and oxygen. With most aromatic substrates, no uncoupling of hydroxylation occurs. Hydroxylation of monofluorinated phenols is highly regiospecific with a preference for C6 hydroxylation. Binding of phenol highly stimulates the rate of flavin reduction by NADPH. At pH 7.6, 25 degrees C, this step does not limit the rate of overall catalysis. During purification, hydroquinone hydroxylase is susceptible towards limited proteolysis. Proteolytic cleavage does not influence the enzyme dimeric nature but results in relatively stable protein fragments of 55, 43, 35 and 22 kDa. N-Terminal peptide sequence analysis revealed the presence of two nick 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.

The ascomycetous yeast Candida parapsilosis CBS604 catabolizes 4-hydroxybenzoate through the initial formation of hydroquinone (1,4-dihydroxybenzene). High levels of hydroquinone hydroxylase activity are induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, 1,3-dihydroxybenzene or 1,4-dihydroxybenzene as the sole carbon source. The monooxygenase constitutes up to 5% of the total amount of protein and is purified to apparent homogeneity in three chromatographic steps. Hydroquinone hydroxylase from C. parapsilosis is a homodimer of about 150 kDa with each 76-kDa subunit containing a tightly noncovalently bound FAD. The flavin prosthetic group is quantitatively resolved from the protein at neutral pH in the presence of chaotropic salts. The apoenzyme is dimeric and readily reconstituted with FAD.Hydroquinone hydroxylase from C. parapsilosis catalyzes the ortho-hydroxylation of a wide range of monocyclic phenols with the stoichiometric consumption of NADPH and oxygen. With most aromatic substrates, no uncoupling of hydroxylation occurs. Hydroxylation of monofluorinated phenols is highly regiospecific with a preference for C6 hydroxylation. Binding of phenol highly stimulates the rate of flavin reduction by NADPH. At pH 7.6, 25 8C, this step does not limit the rate of overall catalysis.During purification, hydroquinone hydroxylase is susceptible towards limited proteolysis. Proteolytic cleavage does not influence the enzyme dimeric nature but results in relatively stable protein fragments of 55, 43, 35 and 22 kDa. N-Terminal peptide sequence analysis revealed the presence of two nick 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.

In an attempt to develop a high producing mammalian cell line expressing CNTO736, a Glucagon like peptide-1-antibody fusion protein (also known as a Glucagon like peptide-1 MIMETIBODY), we have noted that the N-terminal GLP-1 portion of the MIMETIBODY was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. Therefore, a number of parameters that had an effect on productivity as well as product quality were examined. Results suggest that the choice of the host cell line had a significant effect on the overall product quality. Product expressed in mouse myeloma host cell lines had a lesser degree of proteolytic degradation and variability in O-linked glycosylation as compared to that expressed in CHO host cell lines. The choice of a specific CHOK1SV derived clone also had an effect on the product quality. In general, molecules that exhibited minimal N-terminal clipping had increased level of O-linked glycosylation in the linker region, giving credence to the hypothesis that O-linked glycosylation acts to protect against proteolytic degradation. Moreover, products with reduced potential for N-terminal clipping had longer in vivo serum half-life. These findings suggest that early monitoring of product quality should be an essential part of production cell line development and therefore, has been incorporated in our process of cell line development for this class of molecules.