Enzyme Activity Measurement for 4-Hydroxybenzoate 3-Monooxygenase

With years of experience in the development and optimization of enzymatic assays, Creative Enzymes has become one of the most reliable service providers for enzyme activity measurement in the global market. Our capabilities are extensive, covering all aspects of enzyme activity quantification for a large number of different types of enzymes. Here, Creative Enzymes is proud to offer the reliable enzyme activity assay for 4-hydroxybenzoate 3-monooxygenase.

4-Hydroxybenzoate 3-monooxygenase (EC 1.14.13.2; 4-hydroxybenzoate,NADPH:oxygen oxidoreductase (3-hydroxylating)) is the prototype of a large family of pyridine-nucleotide-dependent flavoprotein monooxygenases. This enzyme catalyzes the incorporation of a dioxygen atom into p-hydroxybenzoate (p-OHB) to produce 3,4-dihydroxybenzoate (3,4-DOHB). The 4-hydroxybenzoate 3-monooxygenase-mediated conversion of 4-hydroxybenzoate is a multi-step reaction with three substrates and three products. In the first half-reaction, p-OHB and NADPH bind to the enzyme and NADPH reduces the FAD cofactor. In the ensuing oxidative reactions, reduced flavin reacts with oxygen to form flavin C4a-hydroperoxide, and the distal oxygen of the hydroperoxide is transferred to position 3 of the substrate.

Analysis of the structure-function relationships in 4-hydroxybenzoate 3-monooxygenase has illuminated the role of conformational changes that are important in the catalytic mechanism. Controlled catalysis is achieved by movement of the flavin and protein among three conformations, “in”, “out”, and “open”. The “open” conformation is critical for substrate binding and product release, the “in” conformation for reaction with molecular oxygen and hydroxylation, and the “out” conformation for the reduction of FAD by NADPH. Additionally, it should be noted that the structure of 4-hydroxybenzoate 3-monooxygenase is unusual because there is no recognizable domain for the binding of NADPH involved in the reaction.

Figure: The crystal structure of 4-hydroxybenzoate 3-monooxygenase from Pseudomonas aeruginosa.
PDB: 1K0I

Because the activating mechanism of the reaction catalyzed by 4-hydroxybenzoate 3-monooxygenase is unclear, and the reductive reaction involving NADPH is still unknown, the activity assay of this enzyme could be difficult and sophisticated. To solve this thorny problem, Creative Enzymes offers professional enzyme activity assay of 4-hydroxybenzoate 3-monooxygenase so as to remove any obstacles for its further development and research. We use a reliable spectrophotometric method to determine its enzyme activity by monitoring the consumption of NADPH at 340 nm.

Creative Enzymes is able to work together with our customers to solve specific problems encountered in enzyme activity measurement. Working closely with our customers is integral to our service, and many customers have been loyal to us for many years. Overall, Creative Enzymes is committed to achieving the highest grade of customers’ satisfaction, and to constantly improving its products, services and quality management system.