title: Metabolomics approach for enzyme discovery.
abstract: The search for novel enzymes is an important but difficult task in functional genomics. Here, we present a systematic method based on in vitro assays in combination with metabolite profiling to discover novel enzymatic activities. A complex mixture of metabolites is incubated with purified candidate proteins and the reaction mixture is subsequently profiled by capillary electrophoresis electrospray ionization mass spectrometry (CE-MS). Specific changes in the metabolite composition can directly suggest the presence of an enzymatic activity while subsequent identification of the compounds whose level changed specifically can pinpoint the actual substrate(s) and product(s) of the reaction. We first evaluated the method using several Escherichia coli metabolic enzymes and then applied it to the functional screening of uncharacterized proteins. In this manner, YbhA and YbiV proteins were found to display both phosphotransferase and phosphatase activity toward different sugars/sugar phosphates. Our approach should be broadly applicable and useful for enzyme discovery in any system.
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0017 Japan.
authors.forename: Natsumi
authors.initials: N
authors.lastname: Saito
authors.forename: Martin
authors.initials: M
authors.lastname: Robert
authors.forename: Sayaka
authors.initials: S
authors.lastname: Kitamura
authors.forename: Richard
authors.initials: R
authors.lastname: Baran
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Soga
authors.forename: Hirotada
authors.initials: H
authors.lastname: Mori
authors.forename: Takaaki
authors.initials: T
authors.lastname: Nishioka
authors.forename: Masaru
authors.initials: M
authors.lastname: Tomita
mesh_terms: D019075:Electrophoresis, Capillary; D004798:Enzymes / Q000032:analysis*; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D004798:Enzymes; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; C510856:YbiV protein, E coli