title: DNA-Linked Enzyme-Coupled Assay for Probing Glucosyltransferase Specificity.
abstract: Traditional enzyme characterization methods are low-throughput and therefore limit engineering efforts in synthetic biology and biotechnology. Here, we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor enzyme reactions in a high-throughput manner. Throughput is improved by removing the need for protein purification and by limiting the need for liquid chromatography mass spectrometry (LCMS) product detection by linking enzymatic function to DNA modification. We demonstrate the DLEnCA methodology using glucosyltransferases as an illustration. The assay utilizes cell free transcription/translation systems to produce enzymes of interest, while UDP-glucose and T4-β-glucosyltransferase are used to modify DNA, which is detected postreaction using qPCR or a similar means of DNA analysis. OleD and two glucosyltransferases from Arabidopsis were used to verify the assay's generality toward glucosyltransferases. We further show DLEnCA's utility by mapping out the substrate specificity for these enzymes.
authors.affiliation: Department of Biological Engineering, Synthetic Biology Institute, University of California, Berkeley, Berkeley, California 94704, United States.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sukovich
authors.affiliation: Department of Biological Engineering, Synthetic Biology Institute, University of California, Berkeley, Berkeley, California 94704, United States.
authors.forename: Cyrus
authors.initials: C
authors.lastname: Modavi
authors.affiliation: Department of Biological Engineering, Synthetic Biology Institute, University of California, Berkeley, Berkeley, California 94704, United States.
authors.forename: Markus
authors.initials: M
authors.lastname: de Raad
authors.affiliation: Department of Biological Engineering, Synthetic Biology Institute, University of California, Berkeley, Berkeley, California 94704, United States.
authors.forename: Robin N
authors.initials: RN
authors.lastname: Prince
authors.affiliation: Department of Biological Engineering, Synthetic Biology Institute, University of California, Berkeley, Berkeley, California 94704, United States.
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Anderson
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D010940:Plant Proteins / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D004247:DNA; D005964:Glucosyltransferases; C519503:OleD protein, Streptomyces antibioticus
keywords: biochemical assays; enzyme specificity; glycosyltransferase; synthetic biology