Understanding the molecular mechanism underlying the high catalytic activity of p-hydroxybenzoate hydroxylase mutants for producing gallic acid

p-Hydroxybenzoate hydroxylase (PHBH) is a flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (p-OHB) to 3,4-dihydroxybenzoate (3,4-DOHB). PHBH can bind to other benzoate derivatives in addition to p-OHB; however, hydroxylation occurs only on p-OHB. Replacement of Tyr385 with Phe forms a mutant, which enables the production of 3,4,5-trihydroxybenzonate (gallic acid) from 3,4-DOHB, although the catalytic activity of the mutant is quite low. This study reported how the L199V/Y385F double mutant exhibits 4.3-fold higher activity for producing gallic acid than the Y385F single mutant. This improvement in catalytic activity is primarily due to the suppression of a shunt reaction that wastes reduced nicotinamide adenine dinucleotide phosphate (NADPH) by producing H2O2. To further elucidate the molecular mechanism underlying this higher catalytic activity, we performed molecular dynamics simulations and quantum mechanics/molecular mechanics calculations, in addition to determining th...