Open AccessThe 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
Author(s) -
Mark J. S. Kelly,
Linda Ball,
Cornelia Krieger,
Yinan Yu,
Markus Fischer,
Susanne Schiffmann,
Peter Schmieder,
Ronald Kühne,
Wolfgang Bermel,
Adelbert Bacher,
Gerald Richter,
Hartmut Oschkinat
Publication year - 2001
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.231323598
Subject(s) - chemistry , enzyme , active site , ligand (biochemistry) , nuclear magnetic resonance spectroscopy , stereochemistry , biochemistry , escherichia coli , binding site , residue (chemistry) , mutagenesis , structural genomics , protein structure , isotopic labeling , organic chemistry , receptor , mutation , gene
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.