ATPase activity of magnesium chelatase subunit I is required
to maintain subunit D
in vivo
Vanessa Lake
1,2
, Ulf Olsson
2
, Robert D. Willows
1
and Mats Hansson
2
1
Department of Biological Science, Macquarie University, North Ryde, Australia;
2
Department of Biochemistry, Lund University,
Sweden
During biosynthesis of chlorophyll, Mg
2+
is inserted into
protoporphyrin IX by magnesium chelatase. This enzyme
consists of three different subunits of  40, 70 and
140 kDa. Seven barley mutants deficient in the 40 kDa
magnesium chelatase subunit were analysed and it was
found that this subunit is essential for the maintenance of
the 70 kDa subunit, but not the 140 kDa subunit. The
40 kDa subunit has been shown to belong to the family
of proteins called ÔATPases associated with various cellu-
lar activitiesÕ, known to form ring-shaped oligomeric
complexes working as molecular chaperones. Three of the
seven barley mutants are semidominant mis-sense muta-
tions leading to changes of conserved amino acid residues

are generally named ChlI, ChlD and ChlH [1]. The average
molecular masses of BchI/ChlI, BchD/ChlD and BchH/
ChlH are 40, 70 and 140 kDa, respectively. The largest
subunit is red upon purification due to bound protopor-
phyrin IX [2–4] and binding studies of deuteroporphyrin IX
to the H-subunit show a K
d
value of 0.53–1.2 l
M
[5]. The
large subunit has therefore been suggested to be the catalytic
subunit. The exact role of the other two subunits is not
understood. It is known that they form a complex in the
presence of Mg
2+
and ATP [2,3,6,7]. The complex forma-
tion does not require hydrolysis of ATP, as ADP and
nonhydrolysable ATP analogues (but not AMP) allowed
complex formation [8]. It is clear, however, that the overall
magnesium chelatase reaction requires ATP hydrolysis. The
observations are consistent with earlier observations with
pea magnesium chelatase where the magnesium chelatase
reaction was demonstrated to be a two-step reaction,
consisting of an activation step followed by the actual Mg
2+
insertion step [9]. The activation step could proceed with
ATP-c-S, whereas ATP was required for the chelation.
The three-dimensional structure of the Rhodobacter
capsulatus BchI has recently been determined and it was
found to belong to the large family of ÔATPases associated

Tel.: + 46 46 2220105, E-mail:
Abbreviations: AAA
+
proteins, ATPases associated with various
cellular activities.
(Received 1 December 2003, revised 20 February 2004,
accepted 2 April 2004)
Eur. J. Biochem. 271, 2182–2188 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04143.x
the magnesium chelatase reaction mechanism also takes into
account the structural data of the R. capsulatus BchI and
general functional aspects of AAA
+
proteins [10]. In this
model not only the BchI proteins are organized in an AAA
+
hexamer, but also BchD as the amino acid sequence of the
BchD N-terminal half is homologous to BchI. The interac-
tions between the BchI and BchD proteins are suggested to
occur via three b-hairpin elements, which protrude from the
core of the BchI structure and which do not belong to the
traditional structure of an AAA
+
protein. In the double-
ringed BchI-BchD structure the ATPase activity of BchI is
blocked. A conformational transition upon binding to
BchH may bring the integrin I domain of BchD into contact
with the integrin-binding motif of BchH, simultaneously
triggering porphyrin metallation. This would also lead to a
release of the blockade of the ATP-binding site of BchI by
the integrin I domain, triggering ATP hydrolysis [10]. It is

known that the 40 kDa subunit is encoded by Xantha-h,the
70 kDa subunit by Xantha-g and the 140 kDa subunit
by Xantha-f [24]. The mutations are all lethal. Among
the known seven mutant alleles of the Xantha-h gene
encoding the smallest subunit of barley magnesium chelatase
(corresponding to R. capsulatus BchI), four are recessive
(xantha-h
30
, -h
38
, -h
56
and -h
57
) and three are semidominant
(Xantha-h
clo 125
, -h
clo 157
and -h
clo 161
). The homozygous
mutant plants are all yellow and lack chlorophyll. On the
other hand, the heterozygous mutants carrying the recessive
allele are all green and indistinguishable from the wild-type
plants. In contrast the heterozygous plants carrying the
semidominant allele are pale green. It has been shown that
the recessive mutations prevent transcription of the Xantha-h
gene [24], while the semidominant alleles are mis-sense
mutations leading to changes of single amino acid residues

Barley wild-type (cv. Svalo
¨
f’s Bonus) and barley magnesium
chelatase mutants [23] were grown in moist vermiculite at
20 °C in 12 h dark/light cycles for 8 days. Lights were
turned on at 07:00 h. Yellow homozygous mutant leaves
were sorted from green wild-type leaves and put in liquid
nitrogen. Total barley protein was isolated from frozen
leaves according to [27].
Recombinant R. capsulatus BchI and BchD magnesium
chelatase subunits were used in the study. The BchD protein
was expressed as a His-tagged fusion protein. The BchI and
BchD proteins were produced and purified as described
previously [4].
Ni-affinity chromatography
The Ni-affinity chromatography system of Novagen was
used to immobilize the His-tagged BchD. Ni
2+
was bound
to 1 mL HiTrap Ni-affinity columns (Pharmacia). The
wash buffer contained 20 m
M
imidazole instead of the
recommended 60 m
M
. Four separate columns were used for
the interaction analysis of His-tagged BchD with the three
BchI mutant proteins and the BchI wild-type.
Fig. 1. The reaction catalyzed by magnesium chelatase. The insertion
of Mg

Antibodies were produced against truncated His-tagged
versions of the three barley magnesium chelatase subunits
expressed from derivatives of plasmid pET15b. The plas-
mids containing Xantha-f, -g and -h were named pAntF1:1,
pAntG1 and pAntH, respectively. Plasmid pAntF1:1 con-
tains 741 bp of the Xantha-f gene. The produced polypep-
tide corresponds to amino acid residues E541 to E781 of the
full length XAN-F polypeptide of 1381 amino acid residues
(numbered according to [24]). Plasmid pAntG1 has an insert
of 717 bp of genomic Xantha-g DNA and produces
54 residues of the C-terminal half of the XAN-G protein.
TheXAN-GspecificresiduesAVRVGLNAEKSGDVG
RIMIVAITDGRANVSLKKSNDPEAAAASDAPRPST
QELK follow after the His-tag. Plasmid pAntH contains
749 bp of Xantha-h,  70% of the gene. The XAN-H-
specific amino acid sequence of 239 residues starts with
EVMGP after the His-tag and ends with DISTV. The
fusion proteins were produced in Escherichia coli
BL21(DE3) using the inducible T7 RNA polymerase
system [32]. Cells from 1 L cultures were harvested and
lysed by sonication. His-tagged magnesium chelatase
polypeptides were purified from crude cell extracts accord-
ing to Novagen. All buffers used for the purification of the
XAN-G polypeptide had to contain 6
M
urea to prevent
the protein from precipitation. The proteins were desalted
into 10 m
M
Na-phosphate pH 7.4, 150 m

followed by incubation at 42 °C for 50 min and 70 °Cfor
15 min. One microlitre of RNaseH was added and incuba-
ted for 20 min at 37 °C. The synthesized first strand
cDNA was used as template in a PCR amplification, where
Xantha-g-specific primers were utilized. The primers
EXgLp67 (5¢-CGTAGATACAAACTTGTTCTCGGT
AT-3¢) and EXgUp70 (5¢-GCATTTATTCCCTTCCGTG
GAGACT-3¢) are separated by two introns in the chromo-
somal Xantha-g DNA. Therefore a DNA fragment ampli-
fied from genomic DNA is 566 bp, whereas a fragment
amplified from cDNA is 378 bp. The 50 lL reaction
contained 2 lL first strand cDNA, 5 lL10· reaction
buffer, 3 lLMgCl
2
(25 m
M
), 0.5 lLdNTP(20m
M
),
2 lL of each primer (10 l
M
)and0.5lL Taq DNA
polymerase (5 unitsÆlL
)1
). Thirty-five cycles were per-
formed: 94 °C, 30 s; 58 °C, 30 s; 68 °C, 40 s. After the
PCR was completed the DNA fragments were analysed
with agarose-gel electrophoresis and DNA sequencing.
Results
Presence of magnesium chelatase subunits

clo 157
and -h
clo 161
mutants (Fig. 2B), which have
altered amino acid residues in their resulting 40 kDa
protein. No XAN-H could be found in the recessive
xantha-h
30
, -h
38
,-h
56
and -h
57
mutants (Fig. 2B). This is in
agreement with the lack of Xantha-h mRNA in these
mutants [24]. The large 140 kDa XAN-F protein was not
affected by the mutations and was detected in all of the
seven xantha-h mutants (Fig. 2C).
Binding of mutated BchI to BchD
The barley Xantha-h
clo 125
, -h
clo 157
and -h
clo 161
mutations
have been constructed in the orthologous R. capsulatus
40 kDa magnesium chelatase subunit, BchI, in a previous
study [26]. We analysed the ability of these BchI proteins,

MgCl
2
.Themixturewas
addedto10lL His-tagged BchD (5 mgÆmL
)1
in 50 m
M
Tricine/NaOH pH 8.0, 4 m
M
ATP, 4 m
M
dithiothreitol,
15 m
M
MgCl
2
) and left on ice for 90 min. The resulting
110 lL were mixed with 4 mL binding-buffer (20 m
M
Tris/HCl pH 7.9, 0.5
M
NaCl, 4 m
M
ATP, 15 m
M
MgCl
2
)
and loaded on a Ni
2+

andpurewild-typeBchIwereloadedoneachgelto
identify the proteins in the run-through, wash and elute
fractions. The analysis showed that the His-tagged BchD
bound to the column, as His-tagged BchD was only
found in the elute fractions and not, or to very little
extent, in the run-through and wash fractions. The three
BchI proteins with the exchanges D207N, R289K and
L111F, as well as the wild-type BchI, were found in the
run-through fractions and the first wash fractions, but
also in the elute fraction (Fig. 4A). The experiment was
also performed without His-tagged BchD. The various
BchI proteins probably show some affinity to the HiTrap
Ni-affinity column (Fig. 4C). However, as the amount of
BchI in the elute fractions were much higher when His-
tagged BchD was present in the experiment we conclude
that the four different BchI proteins can all bind to His-
tagged BchD. Further experiments showed that the
binding of wild-type BchI to His-tagged BchD was
dependent on Mg
2+
and that ADP, but not AMP, could
be used instead of ATP. The three modified BchI proteins
could also bind to His-tagged BchD when ADP was used
instead of ATP (Fig. 4B).
Presence of
Xantha-g
mRNA in
xantha-h
mutants
A possible explanation for the absence of 70 kDa XAN-G

column. W1, wash fraction 1; W2, wash fraction 2; W3, wash fraction
3; W4, wash fraction 4; E1, elute fraction 1; E2, elute fraction 2; E3,
elute fraction 3. The arrows indicate the His-tagged BchD.
Fig. 2. Western blot analysis. Analysis of magnesium chelatase sub-
units XAN-G (70 kDa; A), XAN-H (40 kDa; B) and XAN-F
(140 kDa; C) in barley wild-type (Wt) and mutants xantha-h
30
,-h
38
,
-h
56
,-h
57
,-h
clo 125
(DN), -h
clo 157
(RK) and -h
clo 161
(LF). The arrows
indicate the XAN-G, XAN-H and XAN-F antigens.
Ó FEBS 2004 ATPase activity is required to maintain subunit D (Eur. J. Biochem. 271) 2185
cDNAwasthenusedinanordinaryPCRamplification
and the resulting DNA fragments were isolated after
agarose gel electrophoresis and analysed by DNA
sequence analysis. The oligonucleotides used as primers
are separated by two introns in the genomic DNA
fragment. The expected size of a DNA fragment amplified
from the cDNA was 378 bp, whereas the size of a DNA

because wild-type levels of XAN-G are found in eight
available barley Xantha-f mutants deficient in the 140 kDa
XAN-F magnesium chelatase subunit [35]. In the xantha-
h
30
, -h
38
, -h
56
and -h
57
mutants the failure to maintain
XAN-G is easily explained by the absence of XAN-H
protein. In the Xantha-h
clo 125
, -h
clo 157
and -h
clo 161
mutants,
however, the lack of XAN-G has to be explained by an
inhibited activity of the deficient XAN-H proteins. Inter-
estingly, the recombinant R. capsulatus BchI proteins with
exchanged amino acid residues orthologous to the Xantha-
h
clo 125
, -h
clo 157
and -h
clo 161

essential for the survival of the 70 kDa subunit in vivo.We
conclude that the ATPase activity of the 40 kDa subunit is
essential for the function of this subunit as a chaperone
and that binding of I to D is not enough to maintain
the D subunit in the cell. Our study suggests that ATP
hydrolysis is important for a mechanistic step after the
formation of an ID complex. This is supported by studies
performed with N-ethylmaleimide-treated 40 kDa ChlI
subunit of Synechocystis [19]. Similarly to the effects of
the barley Xantha-h
clo 125
, -h
clo 157
and -h
clo 161
mutations
studied here, N-ethylmaleimide treatment abolished ATP
hydrolysis and magnesium chelatase activity, but still
allowed complex formation between the 40 and the
70 kDa subunits. It should be noted that there are examples
of AAA
+
proteins that might function without ATP
hydrolysis [12]. On the other hand it has been shown for
several AAA
+
proteins that a significant change of
conformation occurs during the ATP hydrolysis cycle and
it has been suggested that this may be a general feature of
these proteins [12,36–41].

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