Kinetic analysis revealed that Gun4 dramatically enhances the magnesium chelatase reaction, and reduces the threshold Mg2+concentration required for chelatase activity at low substrate c
Trang 1chlorophyll biosynthesis
Mark Shepherd, Samantha McLean and C Neil Hunter
Robert Hill Institute for Photosynthesis and Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, UK
Magnesium chelatase lies at a branch point in
tetrapyr-role biosynthesis where insertion of Mg2+ eventually
results in the production of chlorophyll, or the insertion
of Fe2+ produces heme Magnesium chelatase is
com-prised of three protein subunits, ChlI (38–42 kDa),
ChlD (60–74 kDa) and ChlH (gun5, 140–150 kDa)
(BchIDH in photosynthetic bacteria) [1–4] ChlI is an
AAA+ATPase [5,6], contains a Mg2+binding site [7],
and forms a stable complex with ChlD [8] The third
subunit, ChlH, binds porphyrins [9,10] and presumably
contains the active site for chelation The steady-state
kinetic characterization of magnesium chelatase
quanti-fied the ATP hydrolysis required to complete a catalytic
cycle and revealed a cooperativity with respect to Mg2+,
which has important implications for regulation of
chlo-rophyll biosynthesis [11] The structure of Gun4, a
pro-tein that binds to the tetrapyrrole substrate and product
of the magnesium chelatase, has recently been solved
[12] Kinetic analysis revealed that Gun4 dramatically
enhances the magnesium chelatase reaction, and reduces the threshold Mg2+concentration required for chelatase activity at low substrate concentrations, implying a possible role for this protein in substrate delivery The next step in chlorophyll biosynthesis, catalysed
by magnesium protoporphyrin IX methyltransferase (ChlM in Synechocystis), involves the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to the propionate group on ring C of magnesium proto-porphyrin IX (MgP) to form magnesium protopor-phyrin IX monomethylester (MgPME) Steady-state kinetic assays showed that the reaction proceeds via a random binding mechanism forming a ternary complex [13] Stopped-flow fluorescence studies indicated that
a relatively slow ( 70 s)1) domain reorganization of ChlM alters the conformation of the MgD binding site and precedes rapid (> 600 s)1) substrate binding (Kd 3.36 lm) [14] Rapid quenched-flow analysis showed that a catalytic intermediate is formed and
Keywords
chlorophyll; chelatase; methyltransferase;
gun signalling
Correspondence
M Shepherd, Department of Biochemistry
and Molecular Biology, A222 Life Sciences
Building, Green Street, University of
Georgia, Athens, GA 30602, USA
Fax: +1 706 5427567
Tel: +1 706 5427252
E-mail: shepherd@secsg.uga.edu
(Received 25 February 2005, revised 5 July
2005, accepted 19 July 2005)
doi:10.1111/j.1742-4658.2005.04873.x
Purified recombinant proteins from Synechocystis PCC6803 were used to show that the magnesium chelatase ChlH subunit stimulates magnesium protoporphyrin methyltransferase (ChlM) activity Steady-state kinetics demonstrate that ChlH does not significantly alter the Kmfor the tetrapyr-role substrate However, quenched-flow analysis reveals that ChlH dramat-ically accelerates the formation and breakdown of an intermediate in the catalytic cycle of ChlM In light of the profound effect that ChlH has on the methyltransferase catalytic intermediate, the pre steady-state analysis in the current study suggests that ChlH is directly involved in the reaction chemistry The kinetic coupling between the chelatase and methyltrans-ferase has important implications for regulation of chlorophyll biosynthesis and for the availability of magnesium protoporphyrin for plastid-to-nucleus signalling
Abbreviations
Mg chelatase, magnesium chelatase; MgD, magnesium deuteroporphyrin IX; MgDME, Mg deuteroporphyrin IX monomethyl ester;
MgP, magnesium protoporphyrin IX; MgPME, Mg protoporphyrin IX monomethyl ester; Mops, 4-morpholinepropanesulfonic acid; PIX, protoporphyrin IX; SAH, S-adenosyl- L -homocysteine; SAM, S-adenosyl- L -methionine; Synechocystis, Synechocystis PCC6803.
Trang 2chelatase, but also of the interaction between these
enzymes The coupling of the magnesium chelatase
and MgP methyltransferase steps is not a new idea; in
1962, inhibition of methyltransferase activity by
ethio-nine resulted in the accumulation of coproporphyrin
(rather than MgP) by whole cells of Rhodobacter
sph-aeroides, which suggested a degree of coupling between
the magnesium chelation and methyltransferase steps
This coupling was proposed to take the form of a
multienzyme complex for the conversion of
proto-porphyrin to magnesium protoproto-porphyrin IX
mono-methylester (MgPME) [21] Subsequently, it was shown
that when Escherichia coli cell extracts containing
the magnesium chelatase H subunit of R capsulatus
(BchH) and the corresponding methyltransferase
(BchM) were mixed, stimulation of BchM activity was
observed [22] However, purified ChlH from
Synecho-cystis was subsequently shown to have no effect on
ChlM activity [23] These are important observations,
and the significance of these findings with respect to
the current data is addressed in the Discussion
In this paper we have used purified recombinant
Synechocystis enzymes to demonstrate that ChlH has
a dramatic stimulatory effect on ChlM catalysis
Quenched-flow experiments show that the magnesium
chelatase H subunit markedly enhances ChlM catalysis
by accelerating the formation and breakdown of the
catalytic intermediate, providing a kinetic link between
the first two reactions of chlorophyll biosynthesis, with
the signalling molecule MgP as the common factor
Interactions between the methyltransferase and
mag-nesium chelatase are likely to be crucial in determining
the availability of MgP for both signalling [20] and
biosynthetic roles in the chloroplast
Results
ChlH stimulates the methyltransferase reaction
ChlM (0.2 lm) was assayed in the presence of 50 lm
MgD, 1 mm SAM and varying concentrations of
ChlH, the porphyrin binding subunit of magnesium chelatase MgD was used, instead of MgP, as the centration of this water-soluble analogue may be con-trolled more easily Figure 1 depicts the increase in the catalytic rate of ChlM when the concentration of ChlH
is increased, which implies that either a ChlH–MgD complex is acting as an activated substrate or that ChlH is directly accelerating the reaction chemistry The plot of steady-state rate (vss) against ChlH concen-tration was fitted to a single rectangular hyperbola ChlM assays were performed as previously reported [13], and v vs [MgD] curves were obtained in the pres-ence and abspres-ence of 4 lm ChlH; this concentration of ChlH gives almost maximal stimulation of methyltrans-ferase activity 0.2 lm ChlM was assayed with 1 mm SAM and various concentrations of MgD Figure 2 shows the rate of MgDME evolution (lmÆmin)1Ælm ChlM)1) vs [MgD] in the presence and absence of 4 lm ChlH Both data sets were fitted to single rectangular hyperbolae and apparent Kmvalues were obtained In the presence and absence of ChlH the apparent Km values were 17.3 ± 3.3 lm and 24.3 ± 7.5 lm, res-pectively
The effect of ChlH on the lag phase prior
to product formation Figure 3A shows quenched-flow ChlM assays in the absence and presence of 0.75 lm ChlH Figure 3B shows the evolution⁄ decay of the catalytic intermediate
Fig 1 Augmentation of the methyltransferase reaction by magnes-ium chelatase H subunit (ChlH) A plot of ChlM catalytic rate (l M
min)1Æl M ChlM)1vs ChlH concentration Methyltransferase assays were performed in the presence of varying concentrations of ChlH protein The reaction mixture contained 100 m M Tris pH 7.5,
100 m M glycerol, 0.2 l M ChlM, 20 l M MgD, 1 m M SAM and var-ious concentrations of ChlH Error bars represent the standard errors when estimating the steady-state rate from timepoints in the stopped assay.
Trang 3in the absence and presence of 0.75 lm ChlH, and
Fig 3C depicts a typical chromatogram obtained
dur-ing HPLC analysis of the quenched-flow samples
ChlM, SAM and ChlH (when present) were
preincu-bated (in 100 mm Tris pH 7.5⁄ 100 mm NaCl) in
syr-inge 1, and MgD was preincubated similarly in syrsyr-inge
2 The quench solution used was the same as that in
steady-state assays (acetone⁄ H2O⁄ 33% ammonia
solu-tion, 80 : 20 : 1) In the absence of ChlH, the lag phase
that precedes MgDME evolution is approximately
150 ms The presence of ChlH reduces this lag phase
to approximately 50 ms, and the amplitude of the
burst phase is enhanced approximately fivefold The
evolution⁄ depletion of the putative catalytic
intermedi-ate was also monitored Figure 3B demonstrintermedi-ates that
the when ChlH is absent, the catalytic intermediate
accumulates between 0 and 150 ms with a rate
con-stant of 24.3 ± 4.1 s)1 When ChlH is present, the
concentration of intermediate appears to decrease
immediately, suggesting that its evolution occurs on a
timescale more rapid than the dead time of the
instru-ment (approximately 2 ms) Hence, this process must
occur with a rate constant in excess of 500 s)1 The
rate of intermediate decay (+ ChlH) was fitted to a
single exponential with a rate constant of 31.7 ±
9.5 s)1 The rate constants for product accumulation
(Fig 3A) may be estimated from the rates of
inter-mediate decay (Fig 3B); rate constants for the
accumu-lation of MgDME are 11.8 s)1 (–ChlH) and 31.7 s)1
(+ 2 lm ChlH) All these parameters are summarized
in Table 1
Fig 2 Rate of ChlM catalysis vs magnesium deuteroporphyrin IX
(MgD) concentration in the presence and absence of 4 l M ChlH
(Mg chelatase H subunit) Plots of ChlM catalytic rate (l M Æmin)1
per l M ChlM) vs MgD concentration The concentrations of ChlM
and SAM were fixed at 0.2 l M , and 1 m M , respectively Assays
were performed in the presence (s) and absence (d) of 4 l M ChlH.
KappMgD ¼ 17.3 ± 3.3 l M in the presence of ChlH KappMgD ¼
24.3 ± 7.5 l M in the absence of ChlH.
Fig 3 Quenched-flow ⁄ HPLC analysis of product and intermediate evolution All solutions contained 100 m M Tris pH 7.5, and 100 m M
NaCl Immediately after mixing, concentrations of ChlM, SAM and MgD were fixed at 0.5 l M , 1 m M and 30 l M , respectively This was performed when ChlM and SAM were preincubated in the pres-ence (s) and abspres-ence (d) of 0.75 l M ChlH in syringe 1 Syringe 2 contained only MgD (A) MgDME evolution was followed by integ-rating the peaks at 12.3 min on the HPLC chromatograms for each timepoint (B) The evolution ⁄ depletion of the putative intermediate was followed by integrating the peaks at 12.7 min on the HPLC chromatograms for each timepoint The data for the decay of inter-mediate in the presence of ChlH were fitted to a three-parameter exponential (k ¼ 31.7 ± 9.5 s)1) The evolution of intermediate in the absence of ChlH was characterized by a single exponential (k ¼ 24.3 ± 4.1 s)1) Units are in arbitrary fluorescence units (AU) (C) A typical HPLC chromatogram to show the elution of MgD, MgDME and the catalytic intermediate (Int).
Trang 4The presence of ChlH clearly exerts a dramatic effect
on the methylation of MgD catalysed by ChlM
(Fig 1) These data appear to conflict with previous
work whereby purified ChlH was found to have no
stimulatory effect on ChlM activity [23] However, that
study used a stopped assay where a single timepoint
was taken after 30 min, which misses the much faster
initial rate seen in the current study, the measurement
of which is complete within 8 min Three hypotheses
present themselves: a ChlH–MgD complex is a
pre-ferred substrate for the methyltransferase, ChlH binds
to ChlM as an allosteric effector, or ChlH accelerates
the reaction chemistry directly The concentration
dependence demonstrates that only a small excess of
ChlH over ChlM is required for maximum rate
enhancement (Fig 1) The ChlH concentration at half
the maximal rate is 1.2 ± 0.3 lm, which might
repre-sent the binding constant (KD) for the binding of ChlH
to ChlM
When excess ChlH was present, the apparent KmMgD
(KappMgD) was 17.3 ± 3.3 lm (Fig 2), whereas in the
absence of ChlH, the KappMgD was 24.3 ± 7.5 lm
(Fig 2) The Kdfor ChlM binding to free MgD,
deter-mined by fluorimetric titration, is 2.4 lm [13]
There-fore, if a ChlH–MgD complex is indeed a preferred
substrate for ChlM, the affinity of the
methyltrans-ferase for such a complex does not appear to be
greater than that of free MgD Also, given that ChlH
does not significantly alter the KmMgD, these
observa-tions suggest an alternative role for ChlH in
stimula-ting the methyltransferase reaction
Figure 3 shows that ChlH reduces the lag phase of
MgDME product evolution (Fig 3A) This is
consis-tent with the data in Fig 3B, where the
evolu-tion⁄ depletion of the putative intermediate is
monitored When ChlH is absent, the intermediate
does not reach the exponential decay phase until at
least 150 ms has elapsed, which coincides with the
evo-lution of MgDME [14] When ChlH is present, the
exponential decay phase of the intermediate occurs much earlier (Fig 3B), and intermediate accumulation occurs within the 2 ms dead time of the instrument This dramatic acceleration in formation of the interme-diate by ChlH, as well as reduction in its lifetime, is consistent with the concomitant decrease in lag phase
of product evolution in Fig 3A The accumulation of intermediate in the absence of ChlH was fitted to an exponential with a rate constant of 24.3 ± 4.1 s)1, which compares to 11.9 s)1 with previous work [14] The current value is a better estimate of intermediate accumulation, as the fit in Fig 3B considers only the evolution of intermediate The rate constant for the decay of intermediate in the presence of ChlH (31.7 ± 9.5 s)1) is three times as large as the value recorded in the absence of ChlH (11.8 s)1 [14]) These rate constants can be used to estimate the rates of MgDME accumulation in Fig 3A, which suggests that ChlH elicits a threefold increase in the rate of product accumulation (Table 1) These data demonstrate that ChlH enhances both the accumulation and decay of this reaction intermediate, resulting in a reduction in the lag phase of product accumulation, and an increase
in initial rate of product evolution Furthermore, the presence of ChlH increases the magnitude of the burst phase approximately fivefold (Table 1), which implies that a greater concentration of enzyme is available to bind MgDME As ChlM appears to bind the inter-mediate more transiently, this is likely to yield a higher available concentration of ChlM to bind other mole-cular species in the reaction
ChlH appears to enhance catalysis by accelerating the formation and decay of a catalytic intermediate This is depicted in Scheme 1 One cannot yet pinpoint the exact mode of action of ChlH, although it is possible that ChlH may possess reactive sidechains involved in methyltransferase catalysis Such roles may include the stabilization of the positive charge on the methyl carbon of SAM, or the enhancement of the negative charge on the propionate carboxyl groups of MgD The rate constants quoted in Scheme 1 are all
Trang 5faster than kcat It has previously been proposed that
product release is the slow step in the reaction [14]
This is consistent with the current work, as ChlH does
not enhance kcat
A recent study showed that MgP accumulation
triggered the alleviation of repression of
photosyn-thetic genes in Arabidopsis [20], and MgP is suggested
to be a signal for one of the plastid to nucleus
signal-ling pathways However, the relative catalytic rates of
magnesium chelatase and MgP methyltransferase may
dictate that there is very little free MgP available for
signalling We have estimated that kcat for Mg
chela-tion is 0.8 min)1 [24], whereas kcat for the subsequent
methyltransferase step is estimated to be 3.4 min)1,
and this is in the absence of ChlH [14] This implies
that in vivo, there may be little unbound MgP,
especi-ally when ChlH is present in excess over that required
for Mg chelation, since methyltransferase activity will
be greatly stimulated The discovery that another
pro-tein, Gun4, can both stimulate Mg chelatase and bind
MgP [12,25] adds another layer of complexity to both
the regulation of chlorophyll biosynthesis, and the
availability of MgP for signalling to the nucleus We
suggest that the relative amounts of both Gun4 and
CHLH are crucial factors that regulate both flux
down the early part of the chlorophyll biosynthetic
pathway and the availability of the MgP signalling
molecule It is known that CHLH expression
exhib-its diurnal fluctuations in Antirrhinum, Arabidopsis,
barley and soybean [3,26–28] and that CHLH is
regu-lated by a circadian clock [29] A regulatory
mecha-nism whereby alterations in magnesium chelatase
H subunit levels affect partitioning between the
mag-nesium (chlorophyll) and iron (haem) branches of
tetrapyrrole biosynthesis was proposed by Gibson
et al [26] Our quantitative data reported here extend the influence of ChlH and show for the first time the way in which this protein exerts a strong effect on the next enzyme in the pathway, ChlM Inspection of the ChlH titration in Fig 1 leads to the conclusion that temporal variations in CHLH (ChlH in plants) concentration in vivo may greatly influence the cata-lytic rate of CHLM, the eukaryotic MgP methyl-transferase This has important implications for the coupling between these steps and for the availability
of MgP for signalling Scheme 2 summarizes these conclusions in terms of variation the magnesium che-latase H subunit and its effect on the tetrapyrrole branchpoint and ChlM, but neglects the effect of Gun4 The fact that variations in ChlM activity are accompanied by altered ferrochelatase activity has been shown recently using transgenic approaches [30]
It would be necessary to establish the levels of these proteins in vivo in order to apply the enhancements measured in this study to a more physiologically rele-vant situation
Experimental procedures All pigments were purchased from Porphyrin Products (Logan, UT, USA) The remaining chemicals were pur-chased from Sigma-Aldrich unless otherwise specified
Protein expression and purification
The plasmid pET9a-His6-ChlM [23] was transformed into
E coli BL21 (DE3) cells and the Synechocystis chlM gene was induced for 15 h at 20C using 0.4 mm isopropyl
Scheme 1 The ChlM reaction and the proposed involvement of the magnesium chelatase H subunit The rate constants are described in Table 1 ChlM, ChlH and the catalytic intermediate are abbreviated as E, H, and Int, respectively.
Trang 6thio-b-d-galactoside The cells were harvested at 3000 g at
4C and cells from 2 L of culture were resuspended in
20 mL chilled binding buffer [20 mm citrate⁄ KOH
(pH 5.8), 500 mm NaCl, 500 mm glycerol, 5 mm imidazole]
The cells were disrupted by sonication for 6· 30 s on ice,
and the cell debris was removed at 39 000 g at 4C The
supernatant was loaded at 2 mLÆmin)1 onto a 2.0 cm·
5.0 cm column packed with Chelating Sepharose Fast-Flow
resin (Amersham Biosciences, Uppsala, Sweden) charged
with 50 mm NiSO4 and pre-equilibrated with three column
volumes of binding buffer The column was washed with
10 column volumes of binding buffer and 6 column
vol-umes of binding buffer containing 60 mm imidazole (wash
buffer) to remove any loosely bound contaminants The
His-tagged ChlM was eluted with binding buffer containing
250 mm imidazole (elute buffer) A 50 mL column of P-6
desalting gel (Bio-Rad, Hercules, CA, USA) was
equili-brated with 50 mm citrate⁄ KOH (pH 5.8), 300 mm
gly-cerol, 200 mm NaCl and used to remove imidazole from
the buffer A typical yield was 15 mg protein from a 2 L
culture of E coli
Porphyrin stocks
Porphyrin solutions were freshly prepared by dissolving a
small amount of porphyrin in buffer A more water-soluble
analogue, magnesium deuteroporphyrin (MgD) was used
instead of MgP The presence of detergent in the assay
buf-fer was no longer required Porphyrin concentrations were
determined in 0.1 m HCl using the e398of 433 000 m)1Æcm)1
[31] after Mg2+had been removed from the porphyrin by a
5-min incubation in 1 m acetic acid SAM and
S-adenosyl-l-homocysteine (SAH) stock solutions were prepared daily
in 0.1 m HCl and 0.1 m NaOH, respectively Their
concen-trations were determined using the e256of 15 200 m)1Æcm)1
in 1 m HCl for SAM and e260of 16 000 m)1Æcm)1 at pH 7
for SAH [32]
ChlM assays
Reactions were carried out at 30C in 100 mm Tris
pH 7.5, 100 mm glycerol, 0.2 lm ChlM, and MgD and SAM concentrations as indicated in the figure legends The assay mixtures were incubated at 30C in the absence of SAM for 5 min to allow for thermal equilibration The SAM was added, and 20-lL aliquots were taken every
2 min over a period of 8 min and quenched in 400 lL stop solution (acetone⁄ water ⁄ 33% ammonia solution,
80 : 20 : 1) These aliquots were centrifuged at 20 000 g for
5 min to pellet any aggregated protein Pigments were sep-arated using reversed phase HPLC [13,14] Between 10 and
70 lL of soluble phase, depending on the MgD concentra-tion, was loaded onto a Beckman ODS Ultrasphere column (150· 4.6 mm; CA, USA) The pigments were separated by
a 7-min linear gradient from 0% to 67% solvent B at
2 mLÆmin)1, and then the gradient was paused for a further
5 min for the porphyrins to elute (Solvent A¼ 0.005% tri-ethylamine in water, solvent B¼ acetonitrile) Eluted por-phyrins were detected with a Waters in-line fluorescence detector Excitation and emission wavelengths were
394 ± 5 nm and 580 ± 5 nm, respectively
The peaks that corresponded to MgD obtained in the elution profiles were integrated using Waters Millennium software Known amounts of MgDME were analysed in the same way to produce a standard curve The maximum rate during an assay was taken as the steady-state rate and occurred at the beginning of the reaction
Quenched-flow measurements
Pre-steady state time samples from ChlM-catalysed reac-tions were obtained using a Hi-Tech rapid quenched flow system All solutions contained 100 mm Tris pH 7.5, and
100 mm NaCl The reaction cell was maintained at a
con-presented, although the effects of Gun4 or
varying porphyrin concentrations are not
included.
Trang 7stant temperature of 30C by circulation of water from a
thermostatically controlled water bath (Grant Instruments,
Cambridge, UK) Reactions were quenched in stop solution
(acetone⁄ water ⁄ 33% ammonia solution, 80 : 20 : 1, v ⁄ v ⁄ v),
and the concentrations of MgDME and intermediate were
determined using reversed phase HPLC, as previously
described [13,14] The data obtained was analysed using
nonlinear regression (sigmaplot 8.0), and apparent rate
constants were obtained by fitting the plots to a single
exponential [y¼ y0 + (1-e–bx)]
Acknowledgements
We thank Mark Hoggins for assisting with the data
analysis This research was funded by the
Biotechno-logy and Biological Sciences Research Council, UK
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