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.

Capillary electrophoresis of proteins 2005–2007

“…CE has been used to study properties of sorghum proteins [188], the effect of iron restriction on the composition of the outer membrane of Pseudomonas strains [189], the effect of several antifungal agents on the protein composition in Candida albicans [190], factors affecting soluble-protein expression [191], effect of different hydrolysates of whey proteins on hepatic glutathione content in mice [192], endogenous serine/threonine Akt phosphorylation [193], dynamic unfolding of regulatory subunit of cAMP-dependent protein kinase [194], glycosylation of recombinant human erythropoietin [195], the effect of heat treatment on properties of Lupinis albus proteins [196], cell adaptation to serum-free conditions on murine erythropoietin production [197], apoptotic pathway in higher eukaryotes [198], nonenzymatic glycosylation of human IgG and HSA with glucosamine and glyceraldehyde [199], thiol distribution in low-density lipoprotein apoprotein [200], structural changes in superoxide dismutase [201], posttranslational modifications of collagen [202,203], proteinprotein interactions [204], aptamers recognizing ricin toxin [205], effect of substrates on enzymatic activity of cellulases from Penicillium brasilianum [206], new enzymatic activities [207], and O-glycosylation of proteins [208].…”

Metabolic profiling of serum using Ultra Performance Liquid Chromatography and the LTQ-Orbitrap mass spectrometry system

“…These include gas chromatography-mass spectrometry (GC-MS) [6,[21][22][23], comprehensive GC×GC-MS [24][25][26], liquid chromatography-mass spectrometry (LC-MS) [27][28][29][30] and variants including Ultra Performance Liquid Chromatography (UPLC) [31][32][33], capillary electrophoresis-mass spectrometry (CE-MS) [34][35][36], direct infusion mass spectrometry (DIMS) [37][38][39][40], Fourier transform infra red spectroscopy (FT-IR) and Raman spectroscopy [41][42][43] and NMR spectroscopy [13,[44][45][46]. Of these, mass spectrometry and NMR have played a key role in the development of metabolomics (and the related discipline of metabonomics).…”

Metabolite Profiling Reveals YihU as a Novel Hydroxybutyrate Dehydrogenase for Alternative Succinic Semialdehyde Metabolism in Escherichia coli

“…However, the existence of an equivalent of the GHBDH reaction and an alternative reductive pathway for GABA metabolism in E. coli is still unreported. * This work was supported by a grant-in-aid for scientific research from the We have previously developed a screening method, based on in vitro assays in combination with metabolite profiling by capillary electrophoresis-mass spectrometry (CE-MS), to discover novel enzymatic activities (17). We hereby refer to this method as Metabolic Enzyme and Reaction discovery by Metabolite profile Analysis and reactant IDentification (MERMAID).…”