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Monooxygenases perform chemo-, regioand/or enantioselective oxygenations of organic substrates under mild reaction conditions. These properties and the increasing number of representatives along with effective preparation methods place monooxygenases in the focus of industrial biocatalysis. Mechanistic and structural insights reveal reaction sequences and allow turning them into efficient tools for the production of valuable products. Herein we describe two biocatalytically relevant subclasses of flavoprotein monooxygenases with a close evolutionary relation: subclass A represented by p-hydroxybenzoate hydroxylase (PHBH) and subclass E formed by styrene monooxygenases (SMOs). PHBH family members perform highly regioselective hydroxylations on a wide variety of aromatic compounds. The more recently discovered SMOs catalyze a number of stereoselective epoxidation and sulfoxidation reactions. Mechanistic and structural studies expose distinct characteristics, which provide a promising source for future biocatalyst development.

Background: 3-Hydroxybenzoate 6-hydroxylase (3HB6H) is a flavoprotein monooxygenase involved in the catabolism of aromatic compounds in soil microorganisms. Results: The enzyme crystal structure features natively bound phospholipids and a Tyr-His pair for substrate binding and catalysis. Conclusion: 3HB6H has a peculiar substrate-binding site that uses a bound lipid to help to discriminate between ortho-and para-hydroxylation. Significance: 3HB6H structure uncovers new flavoprotein strategy for regioselective aromatic hydroxylation.

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Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB), the ring-fission fuel of the gentisate pathway. In this study, the kinetics of reduction of the enzyme-bound flavin by NADH was investigated at pH 8.0 using a stopped-flow spectrophotometer, and the data were analyzed comprehensively according to kinetic derivations and simulations. Observed rate constants for reduction of the free enzyme by NADH under anaerobic conditions were linearly dependent on NADH concentrations, consistent with a one-step irreversible reduction model with a bimolecular rate constant of 43 ± 2 M −1 s −1 . In the presence of 3-HB, observed rate constants for flavin reduction were hyperbolically dependent on NADH concentrations and approached a limiting value of 48 ± 2 s −1 . At saturating concentrations of NADH (10 mM) and 3-HB (10 mM), the reduction rate constant is ∼51 s −1 , whereas without 3-HB, the rate constant is 0.43 s −1 at a similar NADH concentration. A similar stimulation of flavin reduction was found for the enzyme− product (2,5-DHB) complex, with a rate constant of 45 ± 2 s −1 . The rate enhancement induced by aromatic ligands is not due to a thermodynamic driving force because E m 0 for the enzyme−substrate complex is −179 ± 1 mV compared to an E m 0 of −175 ± 2 mV for the free enzyme. It is proposed that the reduction mechanism of 3HB6H involves an isomerization of the initial enzyme− ligand complex to a fully activated form before flavin reduction takes place.