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A Simple In Vitro Assay to Measure the Activity of Geranylgeranyl Diphosphate Synthase and Other Short-Chain Prenyltransferases

  • M. Victoria Barja
  • Manuel Rodríguez-ConcepciónEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2083)

Abstract

Most carotenoids are C40 metabolites produced from C20 geranylgeranyl diphosphate (GGPP). The enzymes that produce this precursor, GGPP synthases (GGPPS), are members of the short-chain prenyltransferase (SC-PT) family. SC-PTs are enzymes that catalyze the sequential head-to-tail addition of one or more C5 molecules of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) with the concomitant release of pyrophosphate (PPi). SC-PTs produce linear isoprenyl diphosphates of up to C20 (GGPP) that serve as precursors for many groups of isoprenoids with a wide range of essential biological functions in Eucarya, Bacteria, and Archaea. Enzymatic analysis of SC-PT activity normally requires complex, laborious and expensive methods such as radioactivity-based assays or liquid chromatography–mass spectrometry (LC-MS). Here we describe a fast and inexpensive spectrophotometric protocol for determining the kinetic parameters of SC-PTs in purified enzyme preparations, using an adapted assay for PPi quantification. We developed the method using the Arabidopsis thaliana GGPPS11 enzyme, which produces geranylgeranyl diphosphate for the synthesis of carotenoids in the chloroplast.

Key words

Short-chain prenyltransferases Isoprenyl diphosphate synthases Enzymatic activity Kinetic parameters EnzChek kit Geranylgeranyl diphosphate synthase GGPP Isoprenoids Carotenoids 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • M. Victoria Barja
    • 1
  • Manuel Rodríguez-Concepción
    • 1
    Email author
  1. 1.Centre for Research in Agricultural Genomics (CRAG)CSIC-IRTA-UAB-UBBarcelonaSpain

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