Metabolomics approach for enzyme discovery.
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.
Saito
Natsumi
N
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0017 Japan.
Robert
Martin
M
Kitamura
Sayaka
S
Baran
Richard
R
Soga
Tomoyoshi
T
Mori
Hirotada
H
Nishioka
Takaaki
T
Tomita
Masaru
M
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
D004798:Enzymes; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; C510856:YbiV protein, E coli