Crystal Structure of 3-Hydroxybenzoate 6-Hydroxylase Uncovers Lipid-assisted Flavoprotein Strategy for Regioselective Aromatic Hydroxylation

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a dimeric flavoprotein that catalyzes the NADH- and oxygen-dependent para -hydroxylation of 3-hydroxybenzoate to 2,5-dihydroxybenzoate. In this study, we report the crystal structure of 3HB6H as expressed in Escherichia coli . The overall fold of 3HB6H is similar to that of p -hydroxybenzoate hydroxylase and other flavoprotein aromatic hydroxylases. Unexpectedly, a lipid ligand is bound to each 3HB6H monomer. Mass spectral analysis identified the ligand as a mixture of phosphatidylglycerol and phosphatidylethanolamine. The fatty acid chains occupy hydrophobic channels that deeply penetrate into the interior of the substrate-binding domain of each subunit, whereas the hydrophilic part is exposed on the protein surface, connecting the dimerization domains via a few interactions. Most remarkably, the terminal part of a phospholipid acyl chain is directly involved in the substrate-binding site. Co-crystallized chloride ion and the crystal structure of the H213S variant with bound 3-hydroxybenzoate provide hints about oxygen activation and substrate hydroxylation. Essential roles are played by His-213 in catalysis and Tyr-105 in substrate binding. This phospholipid-assisted strategy to control regioselective aromatic hydroxylation is of relevance for optimization of flavin-dependent biocatalysts. 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.