Purification and properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel flavin adenine dinucleotide-dependent monooxygenase from Candida parapsilosis CBS604.

摘要: A novel flavoprotein monooxygenase, 4-hydroxybenzoate 1-hydroxylase (decarboxylating), from Candida parapsilosis CBS604 was purified to apparent homogeneity. The enzyme is induced when the yeast grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, or 3,4-dihydroxybenzoate as sole carbon source. monooxygenase a monomer of about 50 kDa containing flavin adenine dinucleotide weakly bound cofactor. 4-Hydroxybenzoate C. catalyzes oxidative decarboxylation wide range derivatives with stoichiometric consumption NAD(P)H and oxygen. Optimal catalysis reached at pH 8, NADH being preferred electron donor. By using (18)O2, it confirmed that oxygen atom inserted into product 1,4-dihydroxybenzene derived molecular 19F nuclear magnetic resonance spectroscopy revealed conversion fluorinated 4-hydroxybenzoates corresponding hydroquinones. activity strongly inhibited by 3,5-dichloro-4-hydroxybenzoate, 4-hydroxy-3,5-dinitrobenzoate, 4-hydroxyisophthalate, which are competitors aromatic substrate. same type inhibition exhibited chloride ions. Molecular orbital calculations show upon deprotonation 4-hydroxy group, nucleophilic reactivity located in all substrates C-1 position. This, fact highly active tetrafluoro-4-hydroxybenzoate 4-hydroxy-3-nitrobenzoate, suggests phenolate forms play an important role catalysis. Based substrate specificity, mechanism proposed for flavin-mediated 4-hydroxybenzoate.