Download Probing the origins of glutathione biosynthesis through biochemical

Biochem. J. (2013) 450, 63–72 (Printed in Great Britain)
63
doi:10.1042/BJ20121332
Probing the origins of glutathione biosynthesis through biochemical
analysis of glutamate-cysteine ligase and glutathione synthetase
from a model photosynthetic prokaryote
William B. MUSGRAVE, Hankuil YI, Dustin KLINE, Jeffrey C. CAMERON, Jonathan WIGNES, Sanghamitra DEY,
Himadri B. PAKRASI and Joseph M. JEZ1
Department of Biology, Washington University, One Brookings Drive, Campus Box 1137, St. Louis, MO 63130, U.S.A.
Glutathione biosynthesis catalysed by GCL (glutamate-cysteine
ligase) and GS (glutathione synthetase) is essential for maintaining redox homoeostasis and protection against oxidative damage
in diverse eukaroytes and bacteria. This biosynthetic pathway
probably evolved in cyanobacteria with the advent of oxygenic
photosynthesis, but the biochemical characteristics of progenitor
GCLs and GSs in these organisms are largely unexplored.
In the present study we examined SynGCL and SynGS from
Synechocystis sp. PCC 6803 using steady-state kinetics. Although
SynGCL shares ∼ 15 % sequence identity with the enzyme from
plants and α-proteobacteria, sequence comparison suggests that
these enzymes share similar active site residues. Biochemically,
SynGCL lacks the redox regulation associated with the plant
enzymes and functions as a monomeric protein, indicating that
evolution of redox regulation occurred later in the green lineage.
Site-directed mutagenesis of SynGCL establishes this enzyme as
part of the plant-like GCL family and identifies a catalytically
essential arginine residue, which is structurally conserved across
all forms of GCLs, including those from non-plant eukaryotes
and γ -proteobacteria. A reaction mechanism for the synthesis of
γ -glutamylcysteine by GCLs is proposed. Biochemical and
kinetic analysis of SynGS reveals that this enzyme shares
properties with other prokaryotic GSs. Initial velocity and product
inhibition studies used to examine the kinetic mechanism of
SynGS suggest that it and other prokaryotic GSs uses a random
ter-reactant mechanism for the synthesis of glutathione. The
present study provides new insight on the molecular mechanisms
and evolution of glutathione biosynthesis; a key process required
for enhancing bioenergy production in photosynthetic organisms.
INTRODUCTION
proteobacteria (e.g. Xanthomonas campestris and Agrobacterium
tumefaciens) form three distinct phylogentic groups, which are
unrelated by sequence, but share common three-dimensional folds
[10–13]. A defining feature of each group is the oligomeric
organization of GCL as a monomeric enzyme in γ - and αproteobacteria, a redox-sensitive heterodimer of catalytic and
regulatory subunits in non-plant eukaryotes, and as a redoxsensitive homodimer in plants [12,14–17]. Similarly, GSs can be
grouped into prokaryotic and eukaryotic forms that function as tetrameric and dimeric enzymes respectively [9,18–23]. Glutathione
biosynthesis and its biological roles in bacteria, yeast, humans and
plants have been well studied, but comparatively little is known
about the enzymes of this pathway in cyanobacteria, which is surprising considering that glutathione metabolism probably evolved
in these organisms with the advent of oxygenic photosynthesis
[9].
In contrast with eukaryotes and microbes, where the
ascorbate/glutathione cycle functions as a central antioxidant system [1,7,8], glutathione alone may act as the primary soluble redox
buffer in cyanobacteria. Cyanobacteria contain high levels of
glutathione that can be further increased by providing exogenous
cysteine [24–27]. The ascorbate levels in cyanobacteria are nearly
250-fold lower than those reported for plant chloroplasts and
genes encoding for ascorbate peroxidase have not been identified
in cyanobacterium, although enzymatic activity has been reported
in Nostoc muscorum 7119 and Synechococcus 6311 and 7942
[25,28,29]. Metabolically, glutathione in Synechocystis is linked
Glutathione (L-γ -glutamyl-L-cysteinyl-glycine) is the central
redox buffer in eukaryotes and many prokaryotes and plays
multiple roles in maintaining cellular homoeostasis in response
to a range of oxidative stresses [1]. As part of these protection
systems, glutathione is critical for redox buffering, detoxification
of xenobiotics, sulfur storage and transport, and as a modifier of
protein function [1–6]. Photosynthetic organisms use multiple
enzymatic and small molecule anti-oxidant and redox-buffering
systems to protect themselves from oxidative damage [7,8];
however, phylogenetic studies suggest that the evolution of
the enzymes required for glutathione biosynthesis arose in
cyanobacteria as protection against ROS (reactive oxygen species)
generated as by-products from photosynthesis and metabolism
[8,9]. The enzymes of the cyanobacterial glutathione biosynthesis
pathway offer a glimpse at the origins of this critical metabolic
system and an understanding of their biochemical properties is
critical in organisms with a potential for sustainable bioenergy
production.
Glutathione synthesis occurs in two sequential ATP-dependent
steps catalysed by GCL (glutamate-cysteine ligase; also known as
γ -glutamylcysteine synthetase; EC 6.3.2.2) and GS (glutathione
synthetase, EC 6.3.2.3) (Figure 1). The GCLs of (i) γ -proteobacteria (e.g. Escherichia coli), (ii) non-plant eukaryotes (e.g.
human, yeast and Drosophila), and (iii) plants (e.g. Arabidopsis
thaliana, Brassica juncea and Physcomitrella patens) and α-
Key words: biosynthesis, cyanobacterium, glutamate-cysteine
ligase (GCL), glutathione, glutathione synthetase (GS),
Synechocystis.
Abbreviations used: BjGCL, Brassica juncea glutamate-cysteine ligase; BSO, buthionine sulfoxime; GCL, glutamate-cysteine ligase; GS, glutathione
synthetase; ROS, reactive oxygen species; SynGCL, Synechocystis sp. PCC 6803 glutamate-cysteine ligase; SynGS, Synechocystis sp. PCC 6803
glutathione synthetase.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2013 Biochemical Society
64
Figure 1
W. B. Musgrave and others
Overall reactions of glutathione biosynthesis
The reactions catalysed by GCL (gshA in Synechocystis sp. PCC6803) and GS (gshB in Synechocystis sp. PCC6803) are shown.
to selenate tolerance [30], iron–sulfur cluster delivery [31,32] and
arsenate tolerance [33].
To date, only limited examinations of GCL and GS, encoded
by the gshA and gshB genes respectively, from Synechocystis and
other cyanobacteria have been reported. Cloning and expression
of the gshA gene from Anabena sp. PCC 7120 confirmed that
it encodes a GCL [34]. Moreover, analysis of a gshA deletion
mutant in Synechocystis sp. PCC 6803 suggested that this gene is
essential for growth, as a homozygous mutant could not be isolated
[35]. A gshB mutant strain was previously isolated from a screen
for pigment biosynthesis in Synechococcus sp. PCC 7942 [36]. In
Synechocystis sp. PCC 6803, generation of a gshB deletion mutant
resulted in accumulation of γ -glutamylcysteine [35]. Additional
studies using the Synechocystis sp. PCC 6803 gshB deletion
mutant established that synthesis of glutathione is beneficial
during acclimation to both environmental and redox perturbations,
especially during extreme oxidative stresses, and is important for
photosynthetic electron transport efficiency, antibiotic resistance
and photosystem stability [35,37,38].
In the present study we examine the biochemical function
of the GCL (SynGCL) and GS (SynGS) from the glutathione
biosynthesis pathway of Synechocystis sp. PCC 6803. Previous
crystallographic studies of the GCL from Indian mustard (B.
juncea) provided information about the active site architecture
of a group 3 enzyme [16], but no functional studies examining
the contributions of active-site residues for either these or any
GCLs have been reported. Identification of amino acid residues
that alter the kinetic properties of SynGCL indicates that this
enzyme shares active site features with the GCL from plants, even
though these enzymes show less than 15 % sequence identity.
Mutagenesis of SynGCL also suggests a common chemical
reaction mechanism for the synthesis of γ -glutamylcysteine in
the first committed step of the pathway in all organisms. Kinetic
analysis of SynGS provides the first examination of the steadystate reaction sequence of a prokaryotic GS. Overall, these
studies reveal the molecular details of the glutathione biosynthesis
pathway in the organism believed to be the evolutionary precursor
of chloroplasts in the green plant lineage. Comparison with
the enzymes from other organisms suggests that biochemical
optimization of these enzymes occurred early with the later
addition of specialized regulatory controls. The generation and
detoxification of ROS is a central factor during many cellular
perturbations with implications in health and biotechnology. Since
glutathione is a key soluble antioxidant and redox buffer, a
mechanistic and kinetic understanding of its synthesis is critical
to understand and modulate cellular redox environment.
EXPERIMENTAL
Materials
E. coli BL21(DE3) cells were purchased from Novagen.
Ni2 + -nitrilotriacetic acid agarose was bought from Qiagen.
c The Authors Journal compilation c 2013 Biochemical Society
Benzamidine–Sepharose and the HiLoad 26/60 Superdex-200
FPLC column were from Amersham. The BioMol Green reagent
used for the detection of inorganic phosphate was purchased
from BioMol Research Laboratories. For the GS assays, γ glutamylcysteine was purchased from Bachem. All other reagents
were purchased from Sigma–Aldrich and were of ACS reagent
quality or better.
Expression construct generation and site-directed mutagenesis
Synechocystis sp. PCC 6803 gshA (slr0990) was PCR-amplified
from the previously described pSL2083 construct [35] with 5 -dTTTCATATGCAATTGACTAAAGGGTTAGAAGT-3 as the forward primer (NdeI site is underlined and start codon is in bold)
and 5 -dATAGTCGACTCACACCAAACTTAGTATTTCATCCCGGGC-3 as the reverse primer (SalI site is underlined and stop
codon is in bold). A similar approach was used for Synechocystis
sp. PCC 6803 gshB (slr1238) from the previously described
pSL2085 construct [35] with 5 -dTTTCATATGAAACTGGCTTTTATTATCGATCC-3 as the forward primer (NdeI site is underlined and start codon is in bold) and 5 -dATAGTCGACCTAAAATTGTTTTTCCAACCAGCAAATTAC-3 as the reverse
primer (SalI site is underlined and stop codon is in bold). The
resulting PCR fragments were digested with NdeI and SalI and
then sub-cloned into pET-28a digested with the same restriction
enzymes to generate the pET-28a-SynGCL and pET-28a-SynGS
constructs. Site-directed mutagenesis of SynGCL used the
®
QuikChange PCR mutagenesis method (Agilent Technologies)
with pET-28a-SynGCL as a template to generate the following
mutants: E37Q, E44Q, T117S, T117A, R167K, R167A, H121Q,
H121A, R248K and R248A.
Protein expression and purification
Protein expression and purification for SynGCL (wild-type and
mutants) and SynGS used similar methods. Expression constructs
were transformed into E. coli BL21(DE3) cells. Transformed E.
coli were grown at 37 ◦ C in Terrific broth containing 50 μg/ml
kanamycin until D600 ≈ 0.8. After induction with 1 mM isopropyl
β-D-thiogalactopyranoside, the cultures were grown at 20 ◦ C for
18 h. Cells were pelleted by centrifugation (4000 g for 10 min)
and resuspended in lysis buffer [50 mM Tris, pH 8.0, 500 mM
NaCl, 25 mM imidazole, 5 mM MgCl2 , 10 % (v/v) glycerol and
1 % (v/v) Tween 20]. After sonication (Fisher Brand Sonicator;
6 × 30 s total time 0.5 s on/off cycle; on ice) and centrifugation
(13 000 g for 30 min), the supernatant was passed over a Ni2 + nitrilotriacetic acid agarose column equilibrated with lysis buffer.
His-tagged protein was eluted with elution buffer (lysis buffer
without Tween 20 and with 250 mM imidazole) and then loaded
on to a Superdex S-200 26/60 size-exclusion FPLC column
equilibrated in 50 mM Hepes, pH 7.5, 5 mM MgCl2 , 100 mM
NaCl and 10 % glycerol. Protein concentration was determined
by the Bradford method (Protein Assay, Bio-Rad Laboratories)
with BSA as standard.
Glutathione biosynthesis in cyanobacteria
Enzyme assays
RESULTS
The enzymatic activities of SynGCL and SynGS were determined
spectrophotometrically at 25 ◦ C by measuring the rate of ADP
formation using a coupled assay with pyruvate kinase and
lactate dehydrogenase [11,21]. A common reaction mixture
(0.5 ml) was used for both proteins and contained 100 mM
Hepes, pH 7.5, 150 mM NaCl, 10 mM MgCl2 , 2 mM sodium
phosphoenolpyruvate, 0.2 mM NADH, 5 units of type III
rabbit muscle pyruvate kinase and 10 units of type II rabbit
muscle lactate dehydrogenase. For SynGCL, standard substrate
concentrations were 1 mM cysteine, 10 mM glutamate and 2 mM
ATP. For SynGS, standard substrate concentrations were 1 mM
γ -glutamylcysteine, 5 mM glycine and 1 mM ATP. Reactions
were initiated by addition of protein and the rate of decrease in
A340 was followed using a Beckman DU800 spectrophotometer.
For determination of the steady-state kinetic parameters, two
substrates were fixed at saturation and the third substrate varied
in concentration and the resulting initial velocity data was fitted
to v = kcat [S]/(K m + [S]) using Kaleidagraph (Synergy Software).
Expression and purification of Synechocystis glutathione
biosynthesis enzymes
Initial velocity analysis of the SynGS kinetic mechanism
Analysis of the kinetic mechanism used global data fitting
analysis [11,21,39]. Reaction rates were measured using standard
assay conditions in a matrix of substrate concentrations (γ glutamylcysteine: 0.05–0.5 mM; ATP: 0.1–1.2 mM; glycine:
0.1–1.2 mM). Global curve fitting in SigmaPlot (Systat
Software) was used for modelling of the kinetic data to rapid
equilibrium rate equations for the 16 possible ter-reactant
kinetic mechanisms [11,21,39]. The best fit of the data was to
v/V max = ([Glu][Cys][ATP]/αβγ K Glu K Cys K ATP )/(1 + [Glu]/K Glu +
[Cys]/K Cys + [ATP]/K ATP + [Glu][Cys]/γ K Glu K Cys + [Glu][ATP]/
β K Glu K ATP + [Cys][ATP]/αK ATP K Cys + [Glu][Cys][ATP]/αβ γ
K Glu K Cys K ATP ), which describes a random, rapid-equilibrium
ter-reactant system.
Product inhibition assays for SynGS
Initial velocities in the presence of either glutathione or phosphate
were monitored spectrophotometrically using the standard assay
system. Enzymatic activity of SynGS was determined after
addition of protein to solutions containing either glutathione (0,
15, 30 and 60 mM) or phosphate (0, 25, 50 and 100 mM) and
various concentrations of γ -glutamylcysteine (0.01–0.15 mM),
glycine (0.1–1 mM) or ATP (0.01–0.3 mM). Assays with added
phosphate used MnCl2 at a 1:1 ratio with ATP in place
of MgCl2 to prevent precipitation of magnesium phosphate.
For assays with added ADP, initial rates were measured by
colorimetric determination of inorganic phosphate. Each 0.1 ml
assay mixture contained 100 mM Hepes, pH 7.5, 150 mM NaCl,
10 mM MgCl2 , various substrate concentrations and ADP (0,
0.25, 0.50, 1 and 2.5 mM). The reaction was initiated by
addition of protein and quenched at several time intervals (0–
5 min) with 1 ml of BioMol Green reagent. Reaction rates were
linear over the time course monitored. Control reactions were
used to correct for background levels of inorganic phosphate
in the reaction mixture. Colour was measured at A620 and
quantified with a standard curve for inorganic phosphate (0–
8 nmol). Global fitting analysis of initial velocity data was
performed with SigmaPlot (Systat Software) using the equations
for competitive inhibition, v = V max /(1 + {(K m /[S])(1 + [I]/K i )});
non-competitive inhibition, v = V max /{(1 + [I]/K i )(1 + K m /[S])};
and uncompetitive inhibition, v = V max /(1 + [I]/K i + K m /[S]).
65
The predicted function of the Synechocystis gshA and gshB
genes as GCL and GS respectively was reported previously
[35]; however, biochemical analysis of these proteins has not
been performed. The nucleotide sequence of Synechocystis gshA
(1170 bp) encodes a 389 amino acid protein with a predicted
molecular mass of 44.1 kDa and a pI value of 5.35 (Figure 2).
Amino acid sequence comparisons show that it shares 64 %
sequence identity with the previously identified GCL from
Anabena [34], 15 % identity with the GCL from A. thaliana
and B. juncea, and less than 12 % identity with the enzyme
from α-proteobacteria (Figure 2). As discussed below, although
the overall similarity between the cyanobacterial and plant GCL
is limited, a number of active site residues described in the
crystal structure of BjGCL (B. juncea GCL) are conserved in
the cyanobacterial enzymes [16]. No significant homology was
detected between the cyanobacterial GCL and the bacterial, fungal
and human enzymes. The nucleotide sequence of Synechocystis
gshB (936 bp) encodes a 320 amino acid protein with a predicted
molecular mass of 35.1 kDa and a pI of 5.39 (Figure 3).
Comparison of SynGS with the enzyme from E. coli revealed
39 % sequence identity, whereas comparison with the GS from
Arabidopsis, human and yeast showed less than 15 % sequence
identity. Active site residues identified in the structure of the E.
coli enzyme in complex with ADP and glutathione are nearly
invariant in SynGS (Figure 3) [40].
For biochemical analysis of the Synechocystis glutathione
biosynthesis enzymes, both were overexpressed in E. coli as
N-terminal hexahistidine-tagged proteins and purified using
nickel-affinity and size-exclusion chromatographies. SDS/PAGE
analysis of the purified recombinant proteins showed that
SynGCL and SynGS migrated with molecular masses of
∼ 46 and ∼ 38 kDa respectively, which corresponded to the
predicted mass of each expressed protein (Supplementary Figure
S1 at http://www.biochemj.org/bj/450/bj4500063add.htm). Gelfiltration chromatography of SynGCL showed that the protein
eluted as a monomeric 45 kDa form, as observed for the
GCL from α- and γ -proteobacteria [12,17]. SynGS eluted
from the size-exclusion column as a 150 kDa species that
represents a tetramer similar to that reported for the E. coli
enzyme [18].
Steady-state kinetic analysis of SynGCL
The steady-state kinetic parameters of the SynGCL for glutamate,
cysteine and ATP were determined using a spectrophotometric
assay (Table 1). The kinetic values were comparable with
those reported for the GCL from Anabena [34]. Enzymatic
activity required the presence of Mg2 + ; however, screening of
other divalent ions (10 mM CaCl2 , 10 mM MnCl2 or 10 mM
CuSO4 ) showed that only Mn2 + could substitute for Mg2 +
with a 5-fold reduction in specific activity. Redox sensitivity in
the plant GCL is mediated by two cysteine residues [11,15–
17], which are absent in the sequences of the cyanobacterial
GCL (Figure 2). Consistent with the lack of both regulatory
cysteine residues in SynGCL, incubation of the protein with either
5 mM 2-mercaptoethanol or dithiothreitol did not alter enzymatic
activity, unlike in plants [11,15–17]. SynGCL was inhibited by
glutathione (IC50 = 7.4 +
− 0.3 mM), which is comparable with the
inhibition of the Arabidopsis and Anabena enzymes [11,34]. BSO
(buthionine sulfoxime), which rapidly inactivates the bacterial
c The Authors Journal compilation c 2013 Biochemical Society
66
Figure 2
W. B. Musgrave and others
Sequence comparison of GCLs from cyanobacteria and plants
Alignment of the sequence of the GCLs from Synechocystis sp. PCC 6803 (SynGCL; accession number slr0990), Anabena sp. PCC 7120 (AnaGCL; accession number NP_487391.1), A. thaliana
(AtGCL; accession number AEE84706.1), B. juncea (BjGCL; accession number CAD91712), P. patens (PpGCL; accession number Phypa1_1:173526), X. campestris (XcGCL; accession number
NP_638742) and A. tumefaciens (AgGCL; accession number NP_353679). For clarity, the alignment does not include the N-terminal regions of the plant and α-proteobacteria sequences that do
not match the cyanobacterial enzymes. Numbering corresponds to SynGCL with every tenth residue marked above the alignment. Invariant residues are highlighted in black with grey text and similar
residues are highlighted in grey with white. Residues that correspond to those in the active site of the BjGCL crystal structure [16] are indicated with an asterisk. Residues in the regulatory disulfide
bond of the plant GCL [15–16] are indicated with a #.
Figure 3
Sequence comparison of GSs from cyanobacteria and bacteria
Alignment of the amino acid sequences of the GSs from Synechocystis sp. PCC 6803 (SynGS; slr1238) and E. coli (EcGS; BAE77010.1). Numbering corresponds to SynGS with every tenth residue
marked above the alignment. Conserved residues are highlighted in grey with white text. Active site residues identified in the E. coli GS crystal structure [40] are indicated with an asterisk.
Table 1
Steady-state kinetic parameters of SynGCL
Active site mutagenesis of SynGCL
Reactions were performed as described in the Experimental section. All V /E tot and K m values
are expressed as a means +
− S.E.M. (n = 3).
Substrate
V /E tot (min − 1 )
K m (μM)
V /K m (M − 1 ·s − 1 )
Glutamate
Cysteine
ATP
21.0 +
− 1.3
25.2 +
− 1.7
30.6 +
− 1.9
953 +
− 16
59 +
− 0.5
145 +
− 13
367
7120
3520
and mammalian enzymes [41,42], is a poor inhibitor of SynGCL
(IC50 = 33.7 +
− 4.7 mM).
c The Authors Journal compilation c 2013 Biochemical Society
Although the cyanobacterial and plant GCLs share limited
sequence identity, alignment of the GCLs from plants,
α-proteobacteria and cyanobacteria shows conservation of
residues across the length of the proteins, including active
site residues identified in the crystal structure of the BjGCL
in complex with the inhibitor BSO [16] (Figures 2 and 4).
In the B. juncea active site (Figure 4), the portion of BSO
corresponding to glutamate is bound by Thr242 , Gln246 , Arg292
and Trp296 , which correspond to Thr117 , His121 , Arg167 and Phe171
in SynGCL. Residues forming the proposed cysteine-binding
site in the plant GCLs (i.e. Arg220 , Tyr221 , Met224 , Met239 , Tyr330
and Phe375 ) are varied in SynGCL (Pro99 , Gln100 , Val116 , Met200
Glutathione biosynthesis in cyanobacteria
Table 2
67
Steady-state kinetic parameters of mutant SynGCL
Reactions were performed as described in the Experimental section. Reactions performed at
sub-saturating (50 mM) glutamate are indicated by an asterisk. V /E tot and K m values were
expressed as a means +
− S.E.M. (n = 3).
Mutant
Substrate
V /E tot (min − 1 )
K m (μM)
V /K m (M − 1 ·s − 1 )
T117S
Glutamate
Cysteine
ATP
Glutamate
Cysteine
ATP
Glutamate
Cysteine*
ATP*
Glutamate
Cysteine*
ATP*
Glutamate
Cysteine
ATP
8.2 +
− 0.6
8.0 +
− 1.3
13.5 +
− 1.5
11.3 +
− 1.4
20.1 +
− 2.7
22.9 +
− 2.2
7.6 +
− 2.4
5.4 +
− 0.1
5.9 +
− 0.2
2.7 +
− 0.7
1.5 +
− 0.2
2.3 +
− 0.3
0.5 +
− 0.1
0.6 +
− 0.1
0.5 +
− 0.1
6100 +
− 150
186 +
− 6.1
352 +
− 19
1180 +
− 135
116 +
− 16
279 +
− 66
15200 +
− 4900
707 +
− 49
310 +
− 18
>50 mM
1950 +
− 180
445 +
− 71
3760 +
− 427
857 +
− 10
969 +
− 77
22.4
717
639
160
2890
1370
8.3
127
317
<0.9
12.8
86.1
2.2
11.7
8.6
H121Q
H121A
R167K
Figure 4
GCL active site
Residues forming the active site of the BjGCL (PDB code 2GWC [16]) are shown. BSO is
labelled with the cysteine (cys) and glutamate (glu) portions of the molecule indicated. The
proposed position of the ATP-binding site in BjGCL is also shown. Residues (in single letter
code) are numbered according to BjGCL with the corresponding residues of SynGCL shown in
parentheses.
and Leu244 ). Two glutamate residues in the Mg2 + -binding site of
BjGCL (Glu159 and Glu165 ) are also found in SynGCL (Glu37 and
Glu44 ). Moreover, an invariant arginine residue (Arg379 in BjGCL;
Arg248 in SynGCL) is positioned between the dipeptide-binding
site and the proposed ATP-binding site in BjGCL.
Due to the low sequence identity between the cyanobacterial
and plant enzymes, we examined the functional contributions of
putative active site residues in SynGCL using a series of sitedirected mutants (E37Q, E44Q, T117S, T117A, H121Q, H121A,
R167K, R167A, R248K and R248A). Each mutant was expressed
in E. coli cells and purified as a soluble monomeric protein,
as determined by size-exclusion chromatography. The SynGCL
E37Q, E44Q, T117A, R167A and R248A mutants displayed no
detectable activity. Other mutants exhibited a range of effects
on the steady-state kinetic parameters (Table 2). The T117S and
H121Q mutants displayed less than 5-fold reductions in turnover
rate and K m values for all three substrates that were increased
3- to 22-fold. Mutation of His121 to an alanine and Arg167 to
a lysine primarily affected the K m value of each mutant for
glutamate, although the cysteine K m value was also strongly
affected. As the concentration for this substrate in assays with
either varied cysteine or varied ATP was not at saturation, the
kinetic parameters of the H121A and R167K mutants should be
considered as estimates. The R248K mutation largely affected the
turnover rate with 40- to 60-fold reductions, along with 15- and 7fold increases in the K m values for cysteine and ATP respectively.
Overall, the effect of mutations in SynGCL were consistent with
the location of these residues in an active site similar to that of
BjGCL and suggest a possible chemical reaction mechanism for
these enzymes.
Steady-state kinetic analysis of SynGS
The steady-state kinetic parameters for γ -glutamylcysteine,
glycine and ATP were determined for SynGS (Table 3). The K m
values of SynGS for the three substrates differ by less than 3-fold
compared with the E. coli enzyme, although the turnover rate of
the cyanobacterial enzyme is ∼ 50-fold slower than the bacterial
GS [43,44]. SynGS is a Mg2 + -dependent enzyme; however, Mn2 +
could substitute with a 2.2-fold reduction in turnover rate at a
10 mM ion concentration. In plants, glutathione analogues with
R248K
Table 3
Steady-state kinetic parameters of SynGS
Reactions were performed as described in the Experimental section. All V /E tot and K m values
were expressed as means +
− S.E.M. (n = 3).
Substrate
V /E tot (min − 1 )
K m (μM)
V /K m (M − 1 ·s − 1 )
γ -Glutamylcysteine
Glycine
ATP
277 +
− 29
253 +
− 35
324 +
− 18
99 +
− 14
407 +
− 90
136 +
− 12
46 630
10 360
39 710
substitutions of the glycine moiety are formed by GS-related
enzymes [23]. To test for possible substrate variation, β-alanine,
serine and glutamate were assayed with SynGS and yielded no
detectable enzymatic activity.
Initial velocity and product inhibition analyses of the kinetic
mechanism of SynGS
To determine the kinetic mechanism of a bacterial GS, initial
velocity studies using SynGS were performed to generate data
in which for a saturating concentration of one substrate the
reaction rates were measured over the entire range of the other
two substrates (Supplementary Figure S2 at http://www.biochemj.
org/bj/450/bj4500063add.htm). The lack of parallel lines in the
double-reciprocal plots eliminated potential ping-pong kinetic
mechanisms from consideration. Global fitting of the initial
velocity data for all three substrates to each of the sixteen rate
equations describing possible ter-reactant systems, which include
six potential ordered mechanisms, nine partially ordered/random
mechanisms and a random kinetic mechanism, yielded a
best fit (r2 = 0.962) using the random ter-reactant equation
[11,21,39]. Analysis using other kinetic mechanism models
yielded unsatisfactory fits (r2 = 0.881–0.920). Fitting of the
data provides estimates of turnover rate, substrate equilibrium
dissociation constants (K ATP , K Gly and K γ EC ), and the interaction
factors between glycine and γ -glutamylcysteine (α), ATP and
γ -glutamylcysteine (β), and ATP and glycine (γ ) (Table 4).
The kinetic parameters determined by global fitting (Table 4),
in which βγ K ATP , αγ K Gly and αβK γ EC correspond to the
apparent K m values for ATP, γ -glutamylcysteine and glycine
respectively, are consistent with those determined by steady-state
kinetic analysis (Table 2). The model-derived parameters suggest
c The Authors Journal compilation c 2013 Biochemical Society
68
W. B. Musgrave and others
Table 4 Kinetic constants for a random ter-reactant model of the SynGS
kinetic mechanism
K ATP , K Gly and K γ EC are the calculated equilibrium dissociation constants for the binding of
substrate with the free enzyme. The interaction factors between glycine and γ -glutamylcysteine
(γ EC), ATP and γ EC, and ATP and glycine are represented by α, β and γ respectively. Values
are means +
− S.E.M.
(i) Fitted parameters
Parameter
Value
V /E tot (min − 1 )
K ATP (μM)
K Gly (μM)
K γ EC (μM)
α
β
γ
184 +
− 25
170 +
− 28
403 +
− 75
211 +
− 33
0.42 +
− 0.12
0.70 +
− 0.20
1.93 +
− 0.37
(ii)Substrate dependences
Kinetic constant
Value
K ATP (μM)
β K ATP
γ K ATP
βγ K ATP
K Gly (μM)
α K Gly
γ K Gly
αγ K Gly
K γ EC (μM)
α K γ EC
β K γ EC
αβ K γ EC
179
125
346
242
403
169
778
1.5
211
89
148
62
Figure 5
Kinetic mechanism of SynGS
A random ter-reactant system best describes the formation of a catalytic ternary complex. K ATP ,
K Gly and K γ EC are the equilibrium constants for substrate binding. Interaction factors between
glycine and γ -glutamylcysteine (γ EC), ATP and γ EC, and ATP and glycine are represented by
α, β and γ respectively. Bold arrows indicate the preferred route to the ternary complex with
broken arrows showing steps with negative interactions between binding sites.
that modest positive and negative interactions occur between
different binding sites. Positive interactions occur between the
ATP and γ -glutamylcysteine (β = 0.70) and the glycine and
γ -glutamylcysteine binding sites (α = 0.42) with a negative
interaction between ATP and glycine (γ = 1.93). The fitted
parameters (Table 4) and the resulting kinetic model (Figure 5)
suggest a random ter-reactant system with a preferred set of
interactions between binding sites for formation of the catalytic
ternary complex in SynGS.
To examine the order of product release from SynGS, the
inhibition patterns for ADP, glutathione and phosphate against
each substrate were analysed (Table 5). ADP was a competitive
c The Authors Journal compilation c 2013 Biochemical Society
Table 5
Product inhibition patterns of SynGS
All assays (n = 3) were performed as described in the Experimental section. γ EC,
γ -glutamylcysteine. The inhibition patterns are as follows: C, competitive; NC, noncompetitive.
Varied substrate
Inhibitor
Fixed substrate
Inhibition
K i (mM)
ATP
γ EC
Glycine
ATP
γ EC
Glycine
ATP
γ EC
Glycine
ADP
ADP
ADP
Glutathione
Glutathione
Glutathione
Pi
Pi
Pi
γ EC (1 mM), glycine (5 mM)
Glycine (5 mM), ATP (2 mM)
γ EC (1 mM), ATP (2 mM)
γ EC (2 mM), glycine (5 mM)
Glycine (5 mM), ATP (2 mM)
γ EC (1 mM), ATP (2 mM)
γ EC (1 mM), glycine (5 mM)
Glycine (5 mM), ATP (2 mM)
γ EC (1 mM), ATP (2 mM)
C
NC
NC
NC
NC
NC
NC
NC
NC
1.12 +
− 0.19
1.41 +
− 0.20
2.19 +
− 0.22
29.9 +
− 5.7
27.3 +
− 6.0
38.6 +
− 3.7
54.0 +
− 5.3
42.8 +
− 7.3
68.2 +
− 9.3
inhibitor with respect to ATP and a non-competitive inhibitor
against both γ -glutamylcysteine and glycine. Both glutathione
and phosphate were non-competitive inhibitors of ATP,
γ -glutamylcysteine and glycine.
DISCUSSION
Although glutathione plays a central role in maintaining
cellular redox homoeostasis, only recently have biochemical and
structural studies provided insight on how the enzymes required
for its biosynthesis function and are regulated. The synthesis
of thiol-containing redox buffers, especially in photosynthetic
organisms, also may have evolved to overcome the instability of
cysteine [43]. The role of GCLs as the first step of glutathione
biosynthesis is thought to have originated in cyanobacteria
and then spread to other bacteria and eukaryotes leading to
three distinct groups of GCLs [9]; however, sequence similarity
between the various types of GCLs is limited. Moreover, little
molecular information on this enzyme from cyanobacteria is
available [34]. Previous bioinformatic analysis showed that the
cyanobacterial GCL share less than 15 % identity with the plant
enzymes and lacked any detectable homology with the GCLs from
γ -proteobacteria (group 1) and non-plant (group 2) eukaryotes.
Interestingly, structure-guided sequence comparison of SynGCL
with the GCLs from A. thaliana and B. juncea (Figures 2 and 4)
suggests that the cyanobacterial and plant GCLs may share similar
active site residues. To investigate this possible relationship,
we first functionally examined SynGCL and then tested the
contribution of conserved residues to catalysis.
Biochemical characterization of SynGCL revealed that the
protein functioned as a monomer, which is similar to that of
the enzyme from the α- and γ -proteobacteria [12,17]. Steadystate kinetic analysis of SynGCL (Table 1) demonstrated that
the cyanobacterial enzyme was insensitive to changes in redox
state, which is consistent with the lack of residues forming
the regulatory disulfide found in the plant GCL [11,15–17].
As reported for the GCLs from E. coli, yeast, A. thaliana
and Anabena [11–13,34], glutathione was a millimolar inhibitor
of SynGCL. The lower K i of glutathione for GCL compared
with GS also suggests that this enzyme is more sensitive to
control by feedback inhibition. Moreover, the poor inhibition of
the cyanobacterial enzyme by BSO, a potent inactivator of the
bacterial and mammalian homologues [41,42], suggests probable
differences in active site structure between the different groups
of GCLs. As the limiting step in the pathway [44], regulation of
GCLs through feedback inhibition by glutathione appears to be
the original control mechanism, with thiol-based redox-regulation
of oligomerization occuring later in plants and the recrutiment of
Glutathione biosynthesis in cyanobacteria
Figure 6
69
Proposed reaction mechanism of SynGCL
a regulatory subunit in the GCLs of non-plant eukaryotes [12,14–
17].
Previous crystallographic studies of the GCL from B. juncea
provided information about the active site architecture of a group
3 enzyme [16] (Figure 4), but no functional studies examining
the contributions of active site residues for these GCL have been
reported. In the BjGCL structure, the position of the inhibitor BSO
delineated residues in proximity to the glutamate- and cysteinederived portions of the ligand and residues forming a Mg2 + binding site [16]. Sequence comparison of the cyanobacterial and
plant GCLs implies that these distantly related enzymes retain a
common active site, as key residues in the active site are invariant
between the two proteins (Figures 2 and 4). On the basis of
the BjGCL structure (Figure 4), Glu37 and Glu44 in SynGCL
would be positioned to bind a catalytically essential Mg2 + ion
and Thr117 , Arg167 and His121 would interact with glutamate. A
role for the invariant arginine residue located at the interface
of the dipeptide- and ATP-binding sites in BjGCL has not been
previously proposed; however, this residue (Arg248 in SynGCL) is
ideally positioned to stabilize the reaction catalysed by the enzyme
(Figure 6). In the present study we used site-directed mutagenesis
to examine the role of invariant residues (i.e. Glu37 , Glu44 , Thr117 ,
Arg167 , His121 and Arg248 ) in SynGCL.
Kinetic analysis of point mutants targeting these residues in
SynGCL suggests that the plant and cyanobacterial GCLs share
common features (Table 2). Mutation of either Glu37 or Glu44 in the
putative Mg2 + -binding site to a glutamine abrogated enzymatic
activity with the side-chain charge being as important to maintain
the position of the metal ion. The effects of the T117S/A,
H121Q/A and R167K/A mutants indicate that these residues,
which are conserved between the plant and cyanobacterial GCL,
are important for efficient formation of γ -glutamylcysteine, most
likely for binding of the glutamate substrate. The T117A and
R167A mutants lacked detectable activity with all the other
mutants exhibiting altered kinetic parameters compared with the
wild-type enzyme. Mutation of His121 to a glutamine residue
yielded less than 3-fold changes in kinetic parameters, whereas
mutation to an alanine residue primarily altered the K m of
glutamate. The T117S mutant displayed a 10- to 15-fold reduction
in catalytic efficiency with all three substrates. The largest changes
were observed with the R167K mutant, which did not show
saturation kinetics using up to 50 mM glutamate. On the basis
of the crystal structure of BjGCL (Figure 4), Arg167 in SynGCL
is predicted to form a critical ionic interaction with the glutamate
backbone carboxy group.
Mutagenesis of Arg248 , which is an invariant residue across
the group 3 GCLs (Figure 2), to an alanine residue results
in an inactive enzyme and the R248K mutation resulted in
an up to 610-fold decrease in V/K m . On the basis of the
position of this arginine residue in the BjGCL crystal structure at
the interface of the putative ATP- and dipeptide-binding sites
and the steady-state kinetic results observed for the SynGCL
R248K and R248A mutants, this residue plays a critical role in
catalysis. The overall reaction catalysed by all GCLs requires
the ATP-dependent phosphorylation of glutamate to form an
acyl-phosphate intermediate. Next, nucleophilic attack of the
cysteine amine group leads to release of inorganic phosphate and
formation of γ -glutamylcysteine [3,11]. We propose a reaction
mechanism for SynGCL (and the other group 3 enzymes) in
which the guanidyl group of Arg248 stabilizes formation of
the pentavalent transition state that results in a phosphorylated
glutamate intermediate and the formation of the transition
state after nucleophilic attack by cysteine (Figure 6). The
catalytically essential Mg2 + , which is co-ordinated by Glu37 and
Glu44 , increases the nucleophilicity of the γ -carboxylate during
formation of the acylphosphate intermediate and stabilizes the
phosphate group in the active site [42,45– 47]. The order of
chemical steps also agrees with the proposed kinetic mechanisms
for the GCLs from A. thaliana and Trypanosoma [11,39].
Although the three groups of GCLs lack sequence homology [10],
the structures of the GCLs from E. coli, yeast and B. juncea reveal
a common three-dimensional fold [12,13,16,47]. Moreover, each
of these enzymes have an arginine residue positioned between
the nucleotide- and amino-acid-binding sites, where this residue
probably plays a similar mechanistic role across all of the GCLs.
Interestingly, the basic features of this reaction are also conserved
across other ATP-dependent peptide synthetases, including GS
[19,20,22,48–50].
GS, the second enzyme in glutathione biosynthesis, has been
extensively studied from eukaryotic sources, including humans,
plants and yeast [8,19–23], and multiple structural studies of
the E. coli GS have been performed [18,40,45,46]; however, no
cyanobacterial GSs have been characterized to date and the kinetic
mechanism of the prokaryotic GSs remains unexamined. Multiple
biochemical features of SynGS, including sequence homology
with E. coli GS (Figure 3), its tetrameric oligomerization and
kinetic properties (Table 3), establish this enzyme as a member of
the prokaryotic GS family.
Analysis of its kinetic mechanism using initial velocity and
product inhibition studies are most consistent with a random
ter-reactant system that displays a preference for the order of
substrate addition. The observed intersection of the initial velocity
patterns (Supplementary Figure S2) indicates that no irreversible
steps occur between the binding of any substrate and removes
c The Authors Journal compilation c 2013 Biochemical Society
70
W. B. Musgrave and others
possible ping-pong type mechanisms from consideration.
These results also suggest the formation of a ternary complex
before catalysis. Global fitting of the initial velocity data to
each of the 16 possible rate equations for ter-reactant kinetic
mechanisms showed that a random mechanism best describes the
observed data (Table 4 and Supplementary Figure S2). The fitted
parameters and substrate dependencies indicate a preference
for substrate addition that leads to the pre-catalytic ternary
complex. The positive interactions between the binding of either
ATP/γ -glutamylcysteine or glycine/γ -glutamylcysteine and the
negative interaction with ATP/glycine suggest a possible kinetic
model for SynGS (Figure 5). In comparison with the kinetic mechanism of A. thaliana GS [21], the substrate dependencies for
the cyanobacterial enzyme are much weaker and may reflect
differences in the active sites of the prokaryotic and eukaryotic
GSs that influence substrate binding.
Product inhibition experiments with SynGS also suggest a
random mechanism (Table 5). If the kinetic mechanism of
SynGS were sequentially ordered, then the observed inhibition
patterns would all be uncompetitive, except for the competitive
inhibition pattern of the last product against the first substrate
[51]. In contrast, a completely random mechanism would lack
any abortive complexes and yield competitive inhibition patterns.
With SynGS, only ADP is a competitive inhibitor compared with
ATP and non-competitive compared with the other two substrates.
These results suggest that both ADP and ATP bind to the same
enzyme form, which suggests that ADP is the last product released
and that ATP is the most likely first substrate to bind. The noncompetitive inhibition of γ -glutamylcysteine and glycine by ADP
indicates that ADP is released from an enzyme form that differs
from the form that binds either substrate. Inhibition by glutathione
and phosphate yielded non-competitive inhibition patterns against
all three substrates and suggested that the reaction products are
either randomly released, form dead-end complexes with free
enzyme and/or an E•S complex, or bind at an allosteric site on
the enzyme [52]. Crystal structures of the E. coli GS [18,40,45,46]
indicate that there is no allosteric binding site present and
suggest that the SynGS active site would not prevent binding
of substrates and products as dead-end complexes. Moreover,
based on the product inhibition data, the release of phosphate
and glutathione from SynGS appears to be random and may
occur before release of ADP. Overall, the initial velocity and
product inhibition profiles are comparable with those described
for multiple other enzymes that use a common set of steps in
their reaction sequences, including the GS from A. thaliana
[21], GCL from A. thaliana and Trypanosoma [11,34], UDPN-acetylmuramate:L-alanine ligase [53], glutamine synthetase
[54], D-alanyl-D-alanine ligase [55] and UDP-N-acetylmuramatetripeptide:D-alanyl-D-alanine-adding enzyme [56]. All of these
enzymes transfer the γ -phosphate group of ATP to a substrate
C-terminal carboxylate followed by nucleophilic attack on the
acylphosphate intermediate by a second substrate leads to
product formation with the release of phosphate, peptide product
and ADP.
The evolution of glutathione biosynthesis in cyanobacteria
coincides with the appearance of photosynthesis. Earlier
bioinformatic analysis suggested that GCL evolved in
cyanobacteria and was later transfered into plants and αproteobacteria [9]; however, the distant homology even among
group 3 GCLs from these organisms made functional comparisons
difficult. The biochemical and mechanistic analyses of SynGCL
for the first time establish a direct link between the GCLs in
cyanobacteria and plants. Although the three distinct groups
of GCLs lack any sequence homology, they share a common
three-dimensional architecture [12,13,16] and, as shown in the
c The Authors Journal compilation c 2013 Biochemical Society
present study, a structurally conserved arginine residue for
catalysis. Similarly, the GSs from prokaryotes and eukaryotes
are two distinct phylogenies [9]; however, both types of
enzymes use a random ter-reactant kinetic mechanism for the
synthesis of a tripeptide critical for protection against oxidative
stresses [21].
Cyanobacteria, such as Synechocystis sp. PCC 6803, are
excellent model systems for understanding photosynthesis and
for testing strategies to enhance its metabolic efficiency for
biofuel applications [57–62]. With engineering and optimization
of oxygenic photosynthesis, alterations in redox environment can
impact electron transport efficiency and photosystem stability and
become a major limiting factor in energy utilization and biofuel
production [59]. Moreover, metabolic engineering for synthesis
of biofuels can also lead to the generation of ROS. For example,
pre-treatment of lignocellulosic biomass for biological conversion
processes releases toxic furan aldehydes, such as furfural, that
promote ROS generation [63]. Similarly, production of biofuel
hexane in Synechocystis sp. PCC 6803 results in increased
expression of enzymes linked to glutathione metabolism [64].
Recent work suggests that enhancing glutathione biosynthesis can
protect against ROS during biofuel production, as the introduction
of GCL and GS into Clostridium acetobutylicum, a microbe
used for butanol production, led to improved survival upon
aeration and increased butanol production [65]. Understanding
the molecular basis of glutathione biosynthesis in cyanobacteria
is an important step toward optimizing these organisms for
future sustainable energy applications and for providing a diverse
repertoire of characterized enzymes with favourable kinetic and
stability properties that might be useful for tailoring the redox
environment of diverse production organisms.
AUTHOR CONTRIBUTION
William Musgrave, Hankuil Yi and Jeffrey Cameron designed the experiments. William
Musgrave, Hankuil Yi, Dustin Kline, Jeffrey Cameron, Jonathan Wignes and Sanghamitra
Dey performed the experiments. William Musgrave, Hankuil Yi, Jeffrey Cameron,
Sanghamitra Dey, Himadri Pakrasi and Joseph Jez wrote the paper.
FUNDING
This work was supported by the National Science Foundation [grant number MCB-0904215
(to J.M.J.)] and the Chemical Sciences, Geosciences and Biosciences Division, Office
of Basic Energy Sciences, Office of Science, U.S. Department of Energy [grant number
DE-FG02-99ER20350 (to H.B.P.)].
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doi:10.1042/BJ20121332
SUPPLEMENTARY ONLINE DATA
Probing the origins of glutathione biosynthesis through biochemical
analysis of glutamate-cysteine ligase and glutathione synthetase
from a model photosynthetic prokaryote
William B. MUSGRAVE, Hankuil YI, Dustin KLINE, Jeffrey C. CAMERON, Jonathan WIGNES, Sanghamitra DEY,
Himadri B. PAKRASI and Joseph M. JEZ1
Department of Biology, Washington University, One Brookings Drive, Campus Box 1137, St. Louis, MO 63130, U.S.A.
Figure S1
SDS/PAGE analysis of SynGCL and SynGS
Samples were stained for total protein using Coomassie Blue. Lanes 1 (MW), 2 (A) and 3 (B)
show the molecular marker (molecular masses in kDa to the left-hand side), purified SynGCL
and purified SynGS respectively.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2013 Biochemical Society
W. B. Musgrave and others
Figure S2
Initial velocity analysis of the kinetic mechanism of SynGS
Experimental data is indicated by the symbols in the reciprocal plots of the initial velocity experiments. The lines shown represent the global fit of all data to the equation for a random ter-reactant
system. Parameters of the fit are listed in Table 4 of the main text. The following conditions were used for each set of assays: (A) 0.5 mM γ -glutamylcysteine (γ EC); 0.12–1.2 mM ATP; and
0.12 (䉱), 0.175 (䊐), 0.35 (䊏), 0.7 (䊊) or 1.2 (䊉) mM glycine; (B) 0.5 mM γ -glutamylcysteine; 0.12–1.2 mM glycine; and 0.12 (䉱), 0.175 (䊐), 0.35 (䊏), 0.7 (䊊) or 1.2 (䊉) mM ATP; (C)
1.2 mM glycine; 0.12–1.2 mM ATP; and 0.05 (䉱), 0.075 (䊐), 0.15 (䊏), 0.3 (䊊) or 0.45 (䊉) mM γ -glutamylcysteine; (D) 1.2 mM glycine; 0.05–0.45 mM γ -glutamylcysteine; and 0.12 (䉱),
0.175 (䊐), 0.35 (䊏), 0.7 (䊊) or 1.2 (䊉) mM ATP; (E) 1.2 mM ATP; 0.05–0.45 mM γ -glutamylcysteine; and 0.12 (䉱), 0.175 (䊐), 0.35 (䊏), 0.7 (䊊) or 1.2 (䊉) mM glycine; and (F) 1.2 mM ATP;
0.12–1.2 mM glycine; and 0.05 (䉱), 0.075 (䊐), 0.15 (䊏), 0.3 (䊊) or 0.45 (䊉) mM γ -glutamylcysteine.
Received 24 August 2012/20 November 2012; accepted 21 November 2012
Published as BJ Immediate Publication 21 November 2012, doi:10.1042/BJ20121332
c The Authors Journal compilation c 2013 Biochemical Society