title: Covalent modification of reduced flavin mononucleotide in type-2 isopentenyl diphosphate isomerase by active-site-directed inhibitors.
abstract: Evidence for an unusual catalysis of protonation/deprotonation by a reduced flavin mononucleotide cofactor is presented for type-2 isopentenyl diphosphate isomerase (IDI-2), which catalyzes isomerization of the two fundamental building blocks of isoprenoid biosynthesis, isopentenyl diphosphate and dimethylallyl diphosphate. The covalent adducts formed between irreversible mechanism-based inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor were investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of IDI-2 binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5, which had been proposed previously. In addition, the high-resolution crystal structures of IDI-2-substrate complexes and the kinetic studies of new mutants confirmed that only the flavin cofactor can catalyze protonation of the substrates and suggest that N5 of flavin is most likely to be involved in proton transfer. These data provide support for a mechanism where the reduced flavin cofactor acts as a general acid/base catalyst and helps stabilize the carbocationic intermediate formed by protonation.
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Nagai
authors.forename: Hideaki
authors.initials: H
authors.lastname: Unno
authors.forename: Matthew Walter
authors.initials: MW
authors.lastname: Janczak
authors.forename: Tohru
authors.initials: T
authors.lastname: Yoshimura
authors.forename: C Dale
authors.initials: CD
authors.lastname: Poulter
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hemmi
mesh_terms: D019748:Carbon-Carbon Double Bond Isomerases / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D002412:Cations; D004583:Electrons; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005486:Flavin Mononucleotide / Q000737:chemistry*; D045782:Hemiterpenes; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D011522:Protons; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D016944:Sulfolobus / Q000378:metabolism
chemical_list: D002412:Cations; D004791:Enzyme Inhibitors; D045782:Hemiterpenes; D011522:Protons; D005486:Flavin Mononucleotide; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase