title: The crystal structure and mechanism of an unusual oxidoreductase, GilR, involved in gilvocarcin V biosynthesis.
abstract: GilR is a recently identified oxidoreductase that catalyzes the terminal step of gilvocarcin V biosynthesis and is a unique enzyme that establishes the lactone core of the polyketide-derived gilvocarcin chromophore. Gilvocarcin-type compounds form a small distinct family of anticancer agents that are involved in both photo-activated DNA-alkylation and histone H3 cross-linking. High resolution crystal structures of apoGilR and GilR in complex with its substrate pregilvocarcin V reveals that GilR belongs to the small group of a relatively new type of the vanillyl-alcohol oxidase flavoprotein family characterized by bicovalently tethered cofactors. GilR was found as a dimer, with the bicovalently attached FAD cofactor mediated through His-65 and Cys-125. Subsequent mutagenesis and functional assays indicate that Tyr-445 may be involved in reaction catalysis and in mediating the covalent attachment of FAD, whereas Tyr-448 serves as an essential residue initiating the catalysis by swinging away from the active site to accommodate binding of the 6R-configured substrate and consequently abstracting the proton of the hydroxyl residue of the substrate hemiacetal 6-OH group. These studies lay the groundwork for future enzyme engineering to broaden the substrate specificity of this bottleneck enzyme of the gilvocarcin biosynthetic pathway for the development of novel anti-cancer therapeutics.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0596, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Noinaj
authors.forename: Mary A
authors.initials: MA
authors.lastname: Bosserman
authors.forename: M Alexandra
authors.initials: MA
authors.lastname: Schickli
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Piszczek
authors.forename: Madan K
authors.initials: MK
authors.lastname: Kharel
authors.forename: Pallab
authors.initials: P
authors.lastname: Pahari
authors.forename: Susan K
authors.initials: SK
authors.lastname: Buchanan
authors.forename: Jürgen
authors.initials: J
authors.lastname: Rohr
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003374:Coumarins / Q000737:chemistry; D018360:Crystallography, X-Ray; D006027:Glycosides / Q000096:biosynthesis* / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
chemical_list: D001426:Bacterial Proteins; D003374:Coumarins; D006027:Glycosides; C031781:gilvocarcin V; D010088:Oxidoreductases