title: Purification and characterization of membrane-bound 3-dehydroshikimate dehydratase from Gluconobacter oxydans IFO 3244, a new enzyme catalyzing extracellular protocatechuate formation.
abstract: 3-Dehydroshikimate dehydratase (DSD) is the first known enzyme catalyzing aromatization from 3-dehydroshikimate (DSA) to protocatechuate (PCA). Differently from cytosolic DSD (sDSD), a membrane-bound 3-dehydroshikimate dehydratase (mDSD) was found for the first time in the membrane fraction of Gluconobacter oxydans IFO 3244, and DSA was confirmed to be the direct precursor of PCA. In contrast to weak and instable sDSD, the abundance of mDSD in the membrane fraction suggested the metabolic significance of mDSD as the initial step in aromatization. mDSD was solubilized only by a detergent and was readily purified to high homogeneity. Its molecular weight was estimated to be 76,000. Purified mDSD showed a sole peak at 280 nm in the absorption spectrum and no critical cofactor requirements. The Km of DSA was measured at 0.5 mM, and the optimum pH was observed at pH 6-8. mDSD appeared to react only with DSA, and was inert to other compounds, such as 3-dehydroquinate, quinate, and shikimate.
authors.affiliation: Department of Chemical and Biological Engineering, Ube National College of Technology, Tokiwadai, Ube, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Shinagawa
authors.forename: Osao
authors.initials: O
authors.lastname: Adachi
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Ano
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Yakushi
authors.forename: Kazunobu
authors.initials: K
authors.lastname: Matsushita
mesh_terms: D000042:Absorption; D000085:Acetates / Q000378:metabolism; D055162:Biocatalysis; D002462:Cell Membrane / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D020600:Gluconobacter oxydans / Q000166:cytology* / Q000201:enzymology; D006836:Hydro-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D062385:Hydroxybenzoates / Q000378:metabolism*; D012995:Solubility
chemical_list: D000085:Acetates; D062385:Hydroxybenzoates; C009091:protocatechuic acid; C044921:3-dehydroshikimate dehydratase; D006836:Hydro-Lyases