Chapter 2
A Simple In Vitro Assay to Measure the Activity
of Geranylgeranyl Diphosphate Synthase and Other
Short-Chain Prenyltransferases
M. Victoria Barja and Manuel Rodrı ´guez-Concepcio ´ n
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
1 Introduction
Isoprenoids (also known as terpenoids) are a widely diverse group
of natural metabolites, and many of them are essential in all-living
organisms. Particularly, plant isoprenoids show an enormous structural and functional diversity. Plants produce a myriad of specialized
isoprenoids involved in their interaction with the environment, but
also a number of essential isoprenoids that participate in photosynthesis (chlorophylls, carotenoids, tocopherols, prenylquinones),
respiration (ubiquinone), or growth regulation (cytokinins, brassinosteroids, gibberellins, abscisic acid, strigolactones) [1]. All
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_2,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
27
A Simple In Vitro Assay to Measure the Activity
of Geranylgeranyl Diphosphate Synthase and Other
Short-Chain Prenyltransferases
M. Victoria Barja and Manuel Rodrı ´guez-Concepcio ´ n
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
1 Introduction
Isoprenoids (also known as terpenoids) are a widely diverse group
of natural metabolites, and many of them are essential in all-living
organisms. Particularly, plant isoprenoids show an enormous structural and functional diversity. Plants produce a myriad of specialized
isoprenoids involved in their interaction with the environment, but
also a number of essential isoprenoids that participate in photosynthesis (chlorophylls, carotenoids, tocopherols, prenylquinones),
respiration (ubiquinone), or growth regulation (cytokinins, brassinosteroids, gibberellins, abscisic acid, strigolactones) [1]. All
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_2,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
27