Crystal structure of 3-hydroxybenzoate 6-hydroxylase uncovers lipid-assisted flavoprotein strategy for regioselective aromatic hydroxylation

J Biol Chem. 2013 Sep 6;288(36):26235-26245. doi: 10.1074/jbc.M113.479303. Epub 2013 Jul 17.


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.

Keywords: Crystal Structure; Flavoproteins; Gentisate; Hydroxylase; Monooxygenase; Oxidation-Reduction; Phospholipid; Rhodococcus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Flavoproteins / chemistry*
  • Flavoproteins / genetics
  • Flavoproteins / metabolism
  • Gene Expression
  • Gentisates / chemistry
  • Gentisates / metabolism
  • Hydroxylation
  • Mass Spectrometry
  • Mixed Function Oxygenases / chemistry*
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism
  • Mutation, Missense
  • NAD / chemistry
  • NAD / genetics
  • NAD / metabolism
  • Phospholipids / chemistry*
  • Phospholipids / genetics
  • Phospholipids / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Rhodococcus / enzymology*
  • Rhodococcus / genetics


  • Bacterial Proteins
  • Flavoproteins
  • Gentisates
  • Phospholipids
  • Recombinant Proteins
  • NAD
  • Mixed Function Oxygenases
  • 3-hydroxybenzoate-6-hydroxylase
  • 2,5-dihydroxybenzoic acid

Associated data

  • PDB/4BJY
  • PDB/4BJZ
  • PDB/4BK1
  • PDB/4BK2
  • PDB/4BK3