title: Mechanistic implications for the chorismatase FkbO based on the crystal structure.
abstract: Chorismate-converting enzymes are involved in many biosynthetic pathways leading to natural products and can often be used as tools for the synthesis of chemical building blocks. Chorismatases such as FkbO from Streptomyces species catalyse the hydrolysis of chorismate yielding (dihydro)benzoic acid derivatives. In contrast to many other chorismate-converting enzymes, the structure and catalytic mechanism of a chorismatase had not been previously elucidated. Here we present the crystal structure of the chorismatase FkbO in complex with a competitive inhibitor at 1.08Å resolution. FkbO is a monomer in solution and exhibits pseudo-3-fold symmetry; the structure of the individual domains indicates a possible connection to the trimeric RidA/YjgF family and related enzymes. The co-crystallised inhibitor led to the identification of FkbO's active site in the cleft between the central and the C-terminal domains. A mechanism for FkbO is proposed based on both interactions between the inhibitor and the surrounding amino acids and an FkbO structure with chorismate modelled in the active site. We suggest that the methylene group of the chorismate enol ether takes up a proton from an active-site glutamic acid residue, thereby initiating chorismate hydrolysis. A similar chemistry has been described for isochorismatases, albeit implemented in an entirely different protein scaffold. This reaction model is supported by kinetic data from active-site variants of FkbO derived by site-directed mutagenesis.
authors.affiliation: Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
authors.forename: Puneet
authors.initials: P
authors.lastname: Juneja
authors.affiliation: Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstr 25, D-79104 Freiburg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Hubrich
authors.affiliation: Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
authors.forename: Kay
authors.initials: K
authors.lastname: Diederichs
authors.affiliation: Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
authors.forename: Wolfram
authors.initials: W
authors.lastname: Welte
authors.affiliation: Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstr 25, D-79104 Freiburg, Germany. Electronic address: jennifer.andexer@pharmazie.uni-freiburg.de.
authors.forename: Jennifer N
authors.initials: JN
authors.lastname: Andexer
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D050505:Mutant Proteins; D007652:Oxo-Acid-Lyases; C075960:chorismate pyruvate lyase
keywords: 3,4-CHD; 3,4-dihydroxycyclohexa-1,5-dienoic acid; 3-HBA; 3-hydroxybenzoic acid; 4-HBA; 4-hydroxybenzoic acid; RidA/YjgF; SeMet; chorismate; high-resolution structure; selenomethionine; site-directed mutagenesis; toblerone fold