title: Crystal structure of 3-hydroxybenzoate 6-hydroxylase uncovers lipid-assisted flavoprotein strategy for regioselective aromatic hydroxylation.
abstract: 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.
authors.affiliation: From the Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands.
authors.forename: Stefania
authors.initials: S
authors.lastname: Montersino
authors.affiliation: Department of Biology and Biotechnology, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy, and.
authors.forename: Roberto
authors.initials: R
authors.lastname: Orru
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Arjan
authors.initials: A
authors.lastname: Barendregt
authors.affiliation: From the Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands.
authors.forename: Adrie H
authors.initials: AH
authors.lastname: Westphal
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Esther
authors.initials: E
authors.lastname: van Duijn
authors.affiliation: Department of Biology and Biotechnology, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy, and. Electronic address: andrea.mattevi@unipv.it.
authors.forename: Andrea
authors.initials: A
authors.lastname: Mattevi
authors.affiliation: From the Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands,. Electronic address: willem.vanberkel@wur.nl.
authors.forename: Willem J H
authors.initials: WJH
authors.lastname: van Berkel
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005841:Gentisates / Q000737:chemistry / Q000378:metabolism; D006900:Hydroxylation; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009243:NAD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005420:Flavoproteins; D005841:Gentisates; D010743:Phospholipids; D011994:Recombinant Proteins; D009243:NAD; D006899:Mixed Function Oxygenases; C023315:3-hydroxybenzoate-6-hydroxylase; C010925:2,5-dihydroxybenzoic acid
keywords: Crystal Structure; Flavoproteins; Gentisate; Hydroxylase; Monooxygenase; Oxidation-Reduction; Phospholipid; Rhodococcus