title: The NMR structure of the 47-kDa dimeric enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase and ligand binding studies reveal the location of the active site.
abstract: Recent developments in NMR have extended the size range of proteins amenable to structural and functional characterization to include many larger proteins involved in important cellular processes. By applying a combination of residue-specific isotope labeling and protein deuteration strategies tailored to yield specific information, we were able to determine the solution structure and study structure-activity relationships of 3,4-dihydroxy-2-butanone-4-phosphate synthase, a 47-kDa enzyme from the Escherichia coli riboflavin biosynthesis pathway and an attractive target for novel antibiotics. Our investigations of the enzyme's ligand binding by NMR and site-directed mutagenesis yields a conclusive picture of the location and identity of residues directly involved in substrate binding and catalysis. Our studies illustrate the power of state-of-the-art NMR techniques for the structural characterization and investigation of ligand binding in protein complexes approaching the 50-kDa range in solution.
authors.affiliation: Research Institute for Molecular Pharmacology, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany. kelly@fmp-berlin.de
authors.forename: M J
authors.initials: MJ
authors.lastname: Kelly
authors.forename: L J
authors.initials: LJ
authors.lastname: Ball
authors.forename: C
authors.initials: C
authors.lastname: Krieger
authors.forename: Y
authors.initials: Y
authors.lastname: Yu
authors.forename: M
authors.initials: M
authors.lastname: Fischer
authors.forename: S
authors.initials: S
authors.lastname: Schiffmann
authors.forename: P
authors.initials: P
authors.lastname: Schmieder
authors.forename: R
authors.initials: R
authors.lastname: Kühne
authors.forename: W
authors.initials: W
authors.lastname: Bermel
authors.forename: A
authors.initials: A
authors.lastname: Bacher
authors.forename: G
authors.initials: G
authors.lastname: Richter
authors.forename: H
authors.initials: H
authors.lastname: Oschkinat
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D008024:Ligands; D019751:Intramolecular Transferases; C066493:L-3,4-dihydroxy-2-butanone-4-phosphate synthase