like native enzyme the arabinoflavin adenine dinucleotide containing 4-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate. The oxidative part of the catalytic cycle of a FAD-containing 4-hydroxybenzoate hydroxylase differs from the native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4-dihydroxybenzoate and 0.7 mol of H2O2 per mol of NADPH oxidized
two flavin conformations in the enzyme: the in-position and the out-position. Substrate hydroxylation occurs while the flavin in the enzyme is in the in-conformation. Flavin must move to the out-conformation for proper formation of the charge-transfer complex between NADPH and FAD that is necessary for rapid flavin reduction
both subunits contain flavin adenine dinucleotide (FAD), which interacts with nicotinamide adenine dinucleotide phosphate (NADPH) and 4-hydroxybenzoate to form a ternary complex that allows the oxygenation of 4-hydroxybenzoate by the reduction of FAD, binding domain structure comparisons, overview
PHBH and related enzymes lack a canonical NAD(P)H-binding domain. The enzyme PHBHCn2 is strictly dependent on NADPH and contains the NADPH-preferring sequence motif 32-EQRSPEYVLGR