Chapter 3
Expression, Purification, and Enzyme Activity Assay
of Phytoene Synthase In Vitro
Maurizio Camagna and Ralf Welsch
Abstract
Phytoene synthase (PSY) is the rate-limiting step in carotenoid biosynthesis, and accordingly subjected to a
number of regulatory mechanisms at various levels, including transcriptional, posttranscriptional, and
posttranslational. Several PSY genes are present in most taxa and show various degrees of tissue and/or
stress-specific responses providing an additional layer of regulating carotenogenesis. Moreover, only a small
number of amino acid differences between paralogs or even single nucleotide polymorphisms distinguishing orthologs greatly affect enzyme properties, suggesting that different enzymatic parameters determined
by intrinsic properties of PSY protein sequences also determine pathway flux. The characterization of
enzyme properties of PSY variants from different origins requires in vitro enzyme assays with recombinant
PSY. In this protocol, we present detailed instructions how to purify several milligrams of active PSY enzyme
from bacterial lysates, which includes initial recombinant PSY enrichment through inclusion body purification, chaotropic unfolding, refolding in presence of detergents and purification through immobilized metal
affinity chromatography. In addition, we provide a protocol to obtain active geranylgeranyl pyrophosphate
(GGPP) synthase as active supply of GGPP substrate is a requirement for high in vitro PSY activity. The
activity assay requires
14 C-labeled substrate and allows to determine its incorporation into phytoene as well
as GGPP. The protocol described here was successfully applied to a variety of PSY and GGPP synthase
homologs from various plant species.
Key words Phytoene synthase, GGPP synthase, Enzyme assay, Protein purification, Protein refolding,
IMAC, Inclusion bodies
1 Introduction
The large diversity of carotenoids results from modifications like
desaturation, cyclization, hydroxylation, and epoxidation reactions
applied to a basic structure. This basic colorless molecule is phytoene, synthesized by the enzyme phytoene synthase (PSY)
through a head-to-head condensation of two molecules geranylgeranyl diphosphate (GGPP). In contrast to the highly lipophilic
phytoene, GGPP represents an amphiphilic prenyl phosphate,
which is also required for the synthesis of other plant compounds
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_3,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
39
Expression, Purification, and Enzyme Activity Assay
of Phytoene Synthase In Vitro
Maurizio Camagna and Ralf Welsch
Abstract
Phytoene synthase (PSY) is the rate-limiting step in carotenoid biosynthesis, and accordingly subjected to a
number of regulatory mechanisms at various levels, including transcriptional, posttranscriptional, and
posttranslational. Several PSY genes are present in most taxa and show various degrees of tissue and/or
stress-specific responses providing an additional layer of regulating carotenogenesis. Moreover, only a small
number of amino acid differences between paralogs or even single nucleotide polymorphisms distinguishing orthologs greatly affect enzyme properties, suggesting that different enzymatic parameters determined
by intrinsic properties of PSY protein sequences also determine pathway flux. The characterization of
enzyme properties of PSY variants from different origins requires in vitro enzyme assays with recombinant
PSY. In this protocol, we present detailed instructions how to purify several milligrams of active PSY enzyme
from bacterial lysates, which includes initial recombinant PSY enrichment through inclusion body purification, chaotropic unfolding, refolding in presence of detergents and purification through immobilized metal
affinity chromatography. In addition, we provide a protocol to obtain active geranylgeranyl pyrophosphate
(GGPP) synthase as active supply of GGPP substrate is a requirement for high in vitro PSY activity. The
activity assay requires
14 C-labeled substrate and allows to determine its incorporation into phytoene as well
as GGPP. The protocol described here was successfully applied to a variety of PSY and GGPP synthase
homologs from various plant species.
Key words Phytoene synthase, GGPP synthase, Enzyme assay, Protein purification, Protein refolding,
IMAC, Inclusion bodies
1 Introduction
The large diversity of carotenoids results from modifications like
desaturation, cyclization, hydroxylation, and epoxidation reactions
applied to a basic structure. This basic colorless molecule is phytoene, synthesized by the enzyme phytoene synthase (PSY)
through a head-to-head condensation of two molecules geranylgeranyl diphosphate (GGPP). In contrast to the highly lipophilic
phytoene, GGPP represents an amphiphilic prenyl phosphate,
which is also required for the synthesis of other plant compounds
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_3,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
39