title: Molecular dynamics simulations and structure-guided mutagenesis provide insight into the architecture of the catalytic core of the ectoine hydroxylase.
abstract: Many bacteria amass compatible solutes to fend-off the detrimental effects of high osmolarity on cellular physiology and water content. These solutes also function as stabilizers of macromolecules, a property for which they are referred to as chemical chaperones. The tetrahydropyrimidine ectoine is such a compatible solute and is widely synthesized by members of the Bacteria. Many ectoine producers also synthesize the stress protectant 5-hydroxyectoine from the precursor ectoine, a process that is catalyzed by the ectoine hydroxylase (EctD). The EctD enzyme is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. A crystal structure of the EctD protein from the moderate halophile Virgibacillus salexigens has previously been reported and revealed the coordination of the iron catalyst, but it lacked the substrate ectoine and the co-substrate 2-oxoglutarate. Here we used this crystal structure as a template to assess the likely positioning of the ectoine and 2-oxoglutarate ligands within the active site by structural comparison, molecular dynamics simulations, and site-directed mutagenesis. Collectively, these approaches suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine. Our study thus provides a view into the catalytic core of the ectoine hydroxylase and suggests an intricate network of interactions between the three ligands and evolutionarily highly conserved residues in members of the EctD protein family.
authors.affiliation: Department of Biology, Laboratory for Microbiology, Philipps-University Marburg, 35037 Marburg, Germany; LOEWE Center for Synthetic Microbiology, Philipps-University Marburg, 35037 Marburg, Germany; Max Planck Institute for Terrestrial Microbiology Emeritus Group R. K. Thauer, 35043 Marburg, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Widderich
authors.affiliation: Department of Biology, Laboratory for Microbiology, Philipps-University Marburg, 35037 Marburg, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Pittelkow
authors.affiliation: X-ray Facility and Crystal Farm, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.
authors.forename: Astrid
authors.initials: A
authors.lastname: Höppner
authors.affiliation: Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mulnaes
authors.affiliation: Department of Biology, Laboratory for Microbiology, Philipps-University Marburg, 35037 Marburg, Germany; LOEWE Center for Synthetic Microbiology, Philipps-University Marburg, 35037 Marburg, Germany; Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buckel
authors.affiliation: Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Gohlke
authors.affiliation: Institute of Biochemistry, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.
authors.forename: Sander H J
authors.initials: SH
authors.lastname: Smits
authors.affiliation: Department of Biology, Laboratory for Microbiology, Philipps-University Marburg, 35037 Marburg, Germany; LOEWE Center for Synthetic Microbiology, Philipps-University Marburg, 35037 Marburg, Germany. Electronic address: bremer@staff.uni-marburg.de.
authors.forename: Erhard
authors.initials: E
authors.lastname: Bremer
mesh_terms: D000599:Amino Acids, Diamino / Q000378:metabolism*; D020134:Catalytic Domain; D007501:Iron / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation
chemical_list: D000599:Amino Acids, Diamino; D007656:Ketoglutaric Acids; C121557:hydroxyectoine; C045628:ectoine; D007501:Iron; D006899:Mixed Function Oxygenases
keywords: DSBH; MD; compatible solute; dioxygenase; double-stranded β-helix; ligand binding; molecular dynamics; molecular modeling; osmotic stress