From the Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, Córdoba 14071, Spain
Received for publication, November 6, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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The molybdenum cofactor (Moco) is essential for
the activity of all molybdoenzymes except nitrogenase. The cDNA for
the Moco carrier protein (MocoCP) of Chlamydomonas
reinhardtii has been cloned by reverse transcription PCR
approaches with primers designed from microsequenced peptides of this
protein. The C. reinhardtii MocoCP has been expressed in
Escherichia coli. The recombinant protein has been
purified to electrophoretic homogeneity and is found assembled into a
homotetramer when Moco is not present under native conditions.
Recombinant MocoCP has the same biochemical characteristics as MocoCP
from C. reinhardtii, as it bound Moco from milk xanthine
oxidase with high affinity, prevented Moco inactivation by oxygen, and
transferred Moco efficiently to aponitrate reductase from the
Neurospora crassa nit1 mutant. The genomic DNA sequence
corresponding to the Chlamydomonas MocoCP gene,
CrMcp1, also was isolated. This gene contained three
introns in the coding region. The deduced amino acid sequence of
CrMcp1 did not show a significant identity to functionally
known proteins in the GenBankTM data base, although a
significant conservation was found with bacterial proteins of unknown
function. The results suggest that proteins having a Moco
binding function probably exist in other organisms.
In eukaryotes, eubacteria, and archaebacteria, all molybdoenzymes
carry Moco1 in their active
sites and share the same core structure. Moco consists of
molybdopterin, an alkylated, not fully aromatic pterin, complexing one
molybdenum atom via a dithiolene group to its four-carbon side chain.
It occurs in several dihydroforms, depending on the enzyme, that
facilitate the oxidation-reduction reaction (1). In prokaryotes, the
cofactor can be modified by an additional nucleotide monophosphate
bound to the C-4' of the phosphate group within the alkylic side
chain of the pterin (2, 3).
Molybdoenzymes are widespread and essential for diverse metabolic
processes such as the first step of nitrate assimilation in autotrophs,
sulfur detoxification and purine catabolism in mammals, and
phytohormone synthesis in plants (4-6). Thus the survival of many
living organisms depends on their ability to synthesize Moco and to
maintain it in a preserved active form. Several diseases have been
related to a defect in Moco synthesis (7). Among the diverse organisms
studied, at least six gene products are involved in the formation of
active Moco (1, 2). Moco biosynthesis occurs by a multistep reaction
of molybdopterin synthesis followed by the incorporation of
molybdenum (2).
Moco activity is routinely assayed by measuring the reconstituted
nitrate reductase activity from the inactive apoenzyme of the
Neurospora crassa fungal mutant strain nit1, which
lacks active Moco (8). This cofactor is very unstable in
vitro, and molybdenum readily dissociates from the cofactor
complex, converting irreversibly into an inactive form such that
molybdate stabilizes Moco (9, 10). Other sulfhydryl-protecting agents
stabilize Moco (10, 11), whereas sulfhydryl-reactive inhibitors prevent
the reconstituting activity of the cofactor (12), indicating the
participation of free sulfhydryl groups in the reconstitution process.
Oxygen seems to be a major factor of free Moco inactivation, which
might be especially dramatic in autotrophs because of the
photosynthetic activity. Thus the presence of a stabilizing
agent in vivo appears to be essential.
Information on the storage of active Moco in the cells is scarce. In a
previous study (13), we purified a Moco-binding protein from
Chlamydomonas reinhardtii. It was named molybdenum cofactor carrier protein (MocoCP) because of its ability to transfer Moco directly to aponitrate reductase (14). This 64-kDa MocoCP
with four identical subunits of 16.5 kDa protected Moco against
inactivation under aerobic conditions and basic pH levels. Proteins
with similar characteristics were also found in Vicia faba
(15) and Escherichia coli (16).
In this work, we present the first molecular evidence for the presence
of a MocoCP in eukaryotes. The recombinant MocoCP binds Moco with a
high affinity and stabilizes Moco against in vitro inactivation by oxygen.
Strains and Growth Conditions--
C. reinhardtii
wild type has been characterized elsewhere (17). Cells were grown at
25 °C under continuous illumination in a liquid minimal medium that
contained 4 mM KNO3 as a nitrogen source
bubbled with 4% (v/v) CO2-enriched air (18). N. crassa nit1 mutant (Fungal Genetic Stock Center, Arcata, CA) was
grown in an ammonium-containing medium and induced in 20 mM nitrate under previously reported conditions (19).
Unless otherwise stated, all E. coli strains were grown in
LB medium supplemented with 50 µg/ml ampicillin, 25 µg/ml
kanamycin, or both. Where appropriate, E. coli strains LE392
and BM25.5 were used for library screening and automatic subcloning,
respectively. Further subcloning of cDNA or genomic DNAs was
performed using E. coli strains DH5 Microsequencing of MocoCP--
MocoCP was purified as reported
previously (13) and subjected to a porous SDS-PAGE (20). The protein
was blotted onto a polyvinylidene difluoride membrane and visualized
with Coomassie Brilliant Blue R-250 stain (0.25% w/v). The
amino-terminal sequence was generated from the protein band cut from
the dry blot. MocoCP was digested with CNBr after gel electrophoresis
(21). The equilibrated gel pieces were subjected to a second
electrophoresis using Tricine-SDS-PAGE (22). The separated peptides
were blotted on a polyvinylidene difluoride membrane and processed like
the amino-terminal blotted protein. Sequencing was carried out at the
Gesellschaft für Biotechnologische Forschung (GBF, Braunschweig,
Germany) on a gas-phase sequencing apparatus. Two sequences were
obtained for peptides from CNBr digestion: GPGKADTAENQLVMANELGKQIATHG
and MGPGTAAEVALALKAKKPVVL.
Oligonucleotide Design, cDNA Synthesis, and Reverse
Transcription PCR--
Two degenerated primers corresponding to
PGKADTAE and MGPGTAAEVA were designed from the microsequenced protein
of purified C. reinhardtii MocoCP by employing the deduced
codon usage for C. reinhardtii genes. These primers are
named MOP1 (forward, 5'-CCCGGCAAGGCCGACCANGCNGAR-3') and MOP2 (reverse,
5'-GCCACCTCGGCGGCGGTNCCNGGNCCCAT-3'), respectively, where N is any of
the four nucleotides and R corresponds to A or G. These primers were
used in reverse transcription PCR for amplification of a MocoCP
cDNA fragment. On the other hand, new primers were designed for
amplifying the complete coding region of the MocoCP gene for expression
in E. coli. New restriction sites for XbaI and
XhoI were included in the forward (MF1,
5'-TCTAGACATGTCGGGACGCAAGCCCATT-3') and reverse (MR1,
5'-CTCGAGCTACGCCAGCAGCTGCTTGAC-3') primers, respectively, to facilitate
further subcloning in the expression vector. The sense primer
contained the start codon in frame with that of glutathione
S-transferase, and the antisense primer contained the stop codon.
cDNA synthesis was performed with 1 µg of total RNA extracted
from the wild type 21gr of C. reinhardtii
cultivated in 4 mM nitrate by reverse transcription
amplification using the SuperscriptTM II kit
(Invitrogen). Reverse transcription PCR was performed with
Taq DNA polymerase (BioTools B&M Laboratories) in reactions containing 2.5% Me2SO, as recommended by the manufacturer,
with the following cycling conditions: 96 °C for 1 min, then 40 cycles at 94 °C for 30 s, 65 °C for 30 s, and 72 °C
for 1 min. The selected PCR fragment was cloned into the pGEM®-T
vector (Promega).
Screenings of C. reinhardtii Libraries and Subcloning--
The
C. reinhardtii cDNA library in DNA Cloning, Sequencing, and Sequence Analyses--
DNA was
sequenced using a dye terminator cycle sequencing ready reaction kit on
an ABI Prism 373 cycle sequencer (PerkinElmer Life Sciences).
DNA sequencing was performed using primers determined by the
vector (T3, T7, SP6, forward or reverse). The nucleotide sequences were
determined in both directions and analyzed using the GCG (Genetics
Computer Group, Madison, WI) and DNASTAR programs. The deduced amino
acid sequence was compared with sequences obtained from searches in the
NCBI Protein Database using the BLASTP algorithm (24). The amino acid
sequences were aligned and converted to a phylogenetic tree using
MegAlign of the DNASTAR package.
Moco and MocoCP Activity--
Active Moco was assayed indirectly
by determining the reconstituted NR activity from extracts of the
N. crassa nit1 mutant as described previously (8). The
extract was prepared as reported previously and frozen at Moco Binding--
Moco binding experiments were performed using
Moco extracted from 250× diluted milk XO as described previously (10).
Freshly isolated Moco (1 µl) was incubated with 0.75 µg of MocoCP
for a defined time at room temperature. Protein-bound and unbound Moco
was separated by gel filtration chromatography with Sephadex G-25 spin
columns (BioRad), and aliquot volumes of the high and low molecular
weight fractions were used for the Nit1 reconstitution assay.
Expression System and Purification of the Recombinant
Protein--
Using insertion mutagenesis PCR, two restriction sites,
XbaI and XhoI, were added to the amplified coding
region of the MocoCP cDNA (495 bp). This DNA was subcloned into
pGEM®-T. The insert fragment was released by digestion with
XbaI/XhoI and subcloned in the expression vector
pGEX-KG (27) in E. coli M-15. The cells from 1-liter
cultures in 2YT medium (23) containing 2% glucose, 50 µg/ml
ampicillin, and 25 µg/ml kanamycin were collected by centrifugation
at an optical density of 1 unit at 600 nm. The cells were washed,
resuspended in 2YT medium containing 50 µg/ml ampicillin, 25 µg/ml
kanamycin, and 200 mM IPTG, and incubated at 37 °C with
shaking for 3 h. The cells were harvested by centrifugation and
resuspended in 10 ml of the PBST extraction buffer (10 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl, 1% Triton
X-100) containing 2 mM EDTA, 0.1% Cloning and Characterization of the MocoCP cDNA and the MocoCP
Gene (CrMcp1) from C. reinhardtii--
A cDNA fragment of 300 bp
was amplified by reverse transcription PCR using the primers MOP1 and
MOP2. They were designed from protein microsequencing of purified
MocoCP from C. reinhardtii and its CNBr digestion peptides.
The DNA sequence of this cDNA fragment included both of the primers
used and additional amino acid residues present in the original protein
microsequencing, which indicated that the correct fragment had been
amplified. This 300-bp fragment was used as a probe to screen a
cDNA library from C. reinhardtii 21gr cells
grown in nitrate medium. From about 80,000 phages screened, 7 hybridizing phages were identified, recovered, and subcloned into the
pEXlox vector. The cloned plasmids released inserts of
0.8-1.0 kb after EcoRI and HindIII digestions. The cDNA sequencing revealed an open reading frame of 495 nucleotides encoding a protein of 165 residues with a calculated
molecular mass of 16.5 kDa. The cDNA also defined 45 nucleotides at the 5'-untranslated region and 446 at the
3'-untranslated region, which has two possible noncanonical and
overlapping polyadenylation signals, TGTCAGTAA (Fig.
1), as compared with the typical signal in Chlamydomonas, and TGTAA (29). A similar situation of
contiguous noncanonical polyadenylation signals was found in the
C. reinhardtii Nar1 gene (30).
The full-length MocoCP cDNA was used as a probe to screen about
60,000 phages from a genomic library in
The comparison of the deduced MocoCP amino acid sequence with
sequences from the GenBankTM showed similarity to proteins
with unknown functions. The sequences with the highest conservation
were from bacteria and Archaea (Fig. 2A). The maximum similarity of
49.7% was found with a putative protein from the cyanobacterium
Trichodesmium erythraeum (GenBankTM
accession number ZP_00073012). Moderate identities were also found with
a putative protein deduced from Aquifex aeolicus (31.1%, GenBankTM accession number AAC06500) and with predicted
Rossmann fold nucleotide-binding proteins from Archaea
Methanopyrus kandleri (33.1%, GenBankTM
accession number NP_614673) and Crenarchaeota 74A4 (27.9%,
GenBankTM accession number AAK96093). A limited and
insignificant identity was found with protein sequences from plants in
the GenBankTM data base. The phylogenetic tree of the
examined proteins (Fig. 2B) shows that the cyanobacterial
protein is grouped together with the C. reinhardtii MocoCP,
both of which are at a similar distance from the proteins from M. kandleri and A. aeolicus, all of which are more
distantly related to the protein from the Crenarchaeota sp.
Although no protein from plants showed conservation with MocoCP, it is
interesting to point out that there is a protein with functionality of
the Moco carrier in V. faba (15).
The molecular analysis of the 165-amino acid sequence of MocoCP using
the program PROTEAN showed that this protein is highly hydrophobic. The
calculated pI of 6.1 is higher than that of 4.5 determined
experimentally for native MocoCP purified from C. reinhardtii (13). This difference could be due to the tetrameric
form of the native protein, which may affect the net charge of the
whole molecule. All examined molybdoenzymes, such as NR from various sources (32, 33), sulfite oxidase of rat liver (34, 35), XO, and
aldehyde oxidase (36), showed an invariant Moco-binding site in the
Moco domain with the characteristic signature sequence CAGNRR. This
sequence is suggested to be involved in Moco binding, and a mutation in
the C residue completely abolishes the NR and sulfite oxidase activity
of Pichia (33, 37). However, the MocoCP-deduced amino acid
sequence does not contain any C residue nor, subsequently, does it
contain the signature sequence CAGNRR. Thus, the Moco-binding site in
MocoCP seems to differ from that in the Moco domain of molybdoenzymes
by allowing both efficient binding and release of Moco.
Southern blot analysis of digested genomic DNA from the C. reinhardtii wild type 6145c using the cDNA as a
probe showed hybridization bands compatible with a single
CrMcp1 gene copy (data not shown).
Little is known about the regulation of Moco genes in eukaryotes. In
plants, Moco gene expression was found to be extremely low so that
standard Northern blots hardly give signals. As expected for
housekeeping genes, Moco genes are expressed constitutively at a basal level (38). The CrMcp1 gene seems to belong to
this family of genes, as it barely gave a signal using standard
Northern blots (data not shown). The availability of sufficient amounts of Moco in a constitutive form is essential for the cell to meet its
changing demand for synthesizing particular enzymes. The multistep synthesis of Moco and the low expression together with the instability of synthesized Moco suggest that the existence of Moco storage proteins
would be a good means by which to buffer the supply and demand of Moco.
In this respect, Chlamydomonas cells contain a much larger
amount of Moco bound to MocoCP than bound to NR (39).
Expression and Functionality of the Recombinant MocoCP in E. coli--
The MocoCP from C. reinhardtii was
expressed in E. coli that had been transformed with the
expression vector pGEX, which contained the full coding region of
MocoCP. Purification of the GST-MocoCP fusion protein had been
performed by specific elution with glutathione in a GSH-agarose
affinity chromatography. After digestion with thrombin, GST and MocoCP
were separated by rechromatography on the same affinity column. MocoCP
eluted directly in the flowing fractions and showed a high homogeneity
as judged by SDS-PAGE (Fig. 3). The
recombinant MocoCP showed a molecular mass higher than that of the
predicted protein (16.5 kDa) given that digestion by thrombin leaves 14 amino acids from the linker region of the fusion protein
(GSPGISGGGGGILD). This glycine-rich linker facilitates the proteolytic
digestion by thrombin (27). As shown below, the recombinant MocoCP has
a high binding affinity to Moco and transfers Moco efficiently to the
apoenzyme from N. crassa nit1 extracts, indicating that this
linker did not affect the function of MocoCP.
In C. reinhardtii, Moco exists in two main forms, bound to
MocoCP and bound to molybdoenzymes, as free Moco is hardly detectable (40). Moco bound to MocoCP in C. reinhardtii is unable to
reconstitute the NR activity of the N. crassa nit1 mutant
when separated by a dialysis membrane, whereas free Moco extracted from
XO can achieve reconstitution. A direct contact between the MocoCP
charged with Moco and the apoNR of N. crassa was proposed
for the reconstitution of the enzyme activity (13, 14). Recombinant
MocoCP was subjected to molecular exclusion chromatography under native
conditions. MocoCP behaved as a protein with a 70-kDa global molecular
mass, which corresponded to a tetramer, whereas the fusion protein
GST-MocoCP appeared to assemble into dimers of 90 kDa and multimers of
more than 280 kDa (data not shown). In the absence of added Moco, both recombinant MocoCP and MocoCP bound to GST did not contain bound Moco
as determined by the Nit1 reconstitution assay (Table I). However, after incubation with Moco from milk XO, recombinant MocoCP
bound Moco but not the MocoCP-GST fusion
protein. These data indicate that Moco is not required for the assembly
of subunits in the quaternary structure of MocoCP and that GST
interferes with the appropriate folding of MocoCP to bind Moco.
The ability of the recombinant MocoCP to bind and protect Moco against
inactivation by oxygen was also assayed by the Nit1 complementation assay. Free Moco was released from milk XO by heat
treatment under anaerobic conditions, and its binding to recombinant
MocoCP was determined at different time intervals under aerobic
conditions after filtering the binding mixtures through a spin column.
Both free Moco and Moco bound to MocoCP were determined in the included
and excluded fractions, respectively. Interestingly,
recombinant MocoCP was able to bind free Moco from XO almost
instantaneously (Table II). Thus
recombinant MocoCP shows an extremely high affinity to bind free Moco
from XO.
Recombinant MocoCP protected Moco against inactivation by oxygen. When
bound to recombinant MocoCP, Moco was stabilized against this
inactivation. Free Moco had lost its activity after 5 h of incubation in aerobic conditions (Fig.
4), whereas Moco bound to recombinant
MocoCP was still more than 93% active under the same conditions. Even
after 72 h in the presence of oxygen, MocoCP conserved 30% of its
Moco activity (Fig. 4). In photosynthetic organisms, large amounts of
oxygen are produced during photosynthesis. This oxygen would inactivate
intracellular free Moco, if present. In C. reinhardtii, the
presence of many light-controlled sites in the promoter of
CrMcp1 would ensure a harmonious induction of this protein
with the production of photosynthetic oxygen. The high affinity binding
of Moco, the low concentration of free Moco, and the necessary contact
of MocoCP and apoNR to transfer Moco (39) together suggest that MocoCP
functions as a binding, storage, protective, and carrier protein. As
with all cellular biomolecules, molybdoenzymes degrade through
normal protein turnover after achieving their catalytic function. There
is no information about the fate of Moco. The low level of Moco
biosynthesis and the high binding affinity of the MocoCP also suggest a
role for MocoCP in the recycling of Moco. Further studies are needed to clarify the role of MocoCP in living organisms.
Conclusions--
The isolation of the cDNA and genomic DNA for
the C. reinhardtii MocoCP provides a definitive molecular
support for the existence of this protein in eukaryotes. The
CrMcp1 gene encodes a protein that contains the sequence of
peptides microsequenced from purified C. reinhardtii MocoCP.
In addition, the protein expressed from cDNA constructs in
E. coli is functionally active and demonstrates the
properties of Moco binding and Moco protection shown for the protein in
the alga. Proteins related to MocoCP from C. reinhardtii appear to be present in cyanobacteria and other bacteria, although a
clear sequence conservation does not appear to exist with other eukaryotic proteins. Our data show that, in
Chlamydomonas, Mcp1 encodes MocoCP, a
homotetrameric protein that binds Moco with a high affinity and plays a
key role in the maintenance and storage of Moco in an active form under
aerobic conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
F' or XL1 blue.
Expression of recombinant MocoCP was carried out in the E. coli strain M-15.
EXlox
(kindly provided by Dr. Paul Lefebvre, University of Minnesota, St.
Paul, MN) was propagated and blotted onto nylon filters. Subsequent
hybridization was performed (23) using a DNA probe randomly labeled by
digoxigenin (Roche Molecular Biochemicals), and positive signals were
detected with alkaline phosphatase-conjugated antidigoxigenin
antibodies and the chemoluminescent substrate CDP-StarTM.
The genomic library in
EMBL4 containing approximate insert sizes of 14 kb (kindly provided by Dr. Carolyn Silflow, University of
Minnesota, St. Paul, MN) was screened, similar to the cDNA library,
by using the MocoCP cDNA as a probe. The inserts were released by
EcoRI digestions and cloned into the pBluescript KS (+/
).
80 °C
until use (13). Moco was released from 250× diluted buttermilk Grade
II (Sigma) XO (EC 1.1.3.22) by heat treatment at 80 °C for 2 min.
The active Moco was determined by reconstitution of the NR activity
(14) after the addition of 1 µl of Moco (in 30 µl total volume)
with or without MocoCP to the apoNR of N. crassa (60 µl).
Then reconstituted NR activity was assayed as reported (25) by
measuring the amount of nitrite produced (26). One unit of active Moco
or MocoCP was defined as the amount of Moco, either free or bound to
MocoCP, that yields one unit of reconstituted NR activity expressed as
the amount of enzyme that catalyzes the reduction of 1 µmol of
nitrate/min.
-mercaptoethanol, and
0.2 mM phenylmethylsulfonyl fluoride. The cells were lysed
by ultrasonication, and the bacterial lysate was centrifuged at
10,000 × g for 10 min to remove the cell debris. The
clear crude extract was applied onto a GSH-agarose column (0.5 × 3 cm) equilibrated with the same extraction buffer. After washing the
matrix with PBST, the fusion protein was eluted with 50 mM
Tris-HCl, pH 8.0, containing 10 mM GSH. The eluted fraction
was concentrated by centrifugation in Centricon (Millipore) and washed
with 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2.5 mM CaCl2, and 0.1% (v/v)
-mercaptoethanol.
50 units of thrombin were added for 4 h at room temperature to
digest the link between GST and MocoCP. The digested protein was
applied onto a minicolumn of Sephadex G-25 to eliminate GSH, and the
high molecular weight fraction was subject to rechromatography in
GSH-agarose. The eluted fractions contained the MocoCP. The purity of
the protein was judged from SDS-PAGE (28) with 15% separation gel
after visualizing the protein bands with Coomassie Brilliant Blue
R-250. The molecular mass was calculated from migrations of molecular
weight markers (Sigma). The concentrations of the purified protein were
determined from the UV light absorption at 280 nm corresponding to the
single tryptophan residue of MocoCP (
= 5.6 M
1 cm
1).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and deduced amino acid sequences
of the CrMcp1 gene. Mcp1 sequence
(GenBankTM accession number AY039706) shows the promoter
region, 4 exons (capital letters) separated by 3 introns
(lowercase letters), and the 3'-untranslated region. Two
putative nitrate-controlled signals are indicated in bold
letters in the promoter sequence. The nucleotide sequence for the
cDNA (GenBankTM accession number AY039707) is shown as
underlined letters at the 5'- and 3'-untranslated regions,
and the coding sequence is shown in capital letters. Two
overlapping polyadenylation signals (bold, underlined
letters) in the 3'-untranslated region also are indicated.
EMBL4. Nine clones with the
highest signals were chosen for further study. These phages
corresponding to the C. reinhardtii MocoCP gene
(CrMcp1) released inserts with an average length of 5-12 kb
by EcoRI digestion as indicated by Southern blot analysis.
One of these fragments of about 6 kb was cloned in Bluescript KS
(+/
) and sequenced. The DNA sequencing of the genomic clone
revealed an open reading frame of 495 nucleotides similar to that of
MocoCP cDNA, which is divided into 4 exons (capital letters)
separated by 3 introns (lowercase letters). The introns are defined by
the typical splicing sites in C. reinhardtii,
5',G-gt(g/a)(a/g)g and 3', (c/t)ag-(A/G/C), (29). The promoter
region (Fig. 1) would cover 600 bp with a putative TATA box (position
240 from the beginning of the cDNA), two
nitrate-dependent transcription motifs (positions
184 and
113) corresponding to an AT-rich sequence followed by (G/A)(C/G)TCA (31), and 10 possible boxes related to light control and described in
plants (sphinx.rug.ac.be:8080/PlantCARE/cgi/index.html). The CrMcp1 gene shows the usual features of C. reinhardtii genes, such as the presence of many introns despite
the small size of the coding region, a long 3'-untranslated region, and
particular polyadenylation sequences.
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Fig. 2.
Amino acid sequence alignment and
phylogenetic tree of MocoCP and potentially related proteins.
A, the deduced amino acid sequence of MocoCP encoded by the
CrMcp1 gene was compared with other sequences of the
GenBankTM data base. The alignment was generated with the
MegAlign program. The protein sequences were derived from the following
organisms, which are provided in the order shown in the figure with the
corresponding GenBankTM accession numbers shown
in parentheses: C. reinhardtii (AY039707), T. erythraeum (ZP_00073012), M. kandleri (NP_614673),
A. aeolicus (AAC06500), and Crenarchaeota 74A4
(AAK96093). B, the phylogenetic tree for MocoCP.
Panels A and B were made from the alignment with
ClustalW using the MegAlign program.
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Fig. 3.
SDS-PAGE analysis for the purification steps
of recombinant MocoCP from C. reinhardtii. After electrophoresis, the gel was
stained by Coomassie R-250. Molecular mass of the markers is indicated
in kDa. a, crude extract IPTG; b, crude
extract + IPTG; c, purified fusion protein (recombinant
MocoCP + GST); d, digested fusion protein; e,
recombinant purified MocoCP; f, GST.
Moco binding activity of recombinant MocoCP from C. reinhardtii
Efficiency of recombinant MocoCP to bind Moco extracted from milk
XO
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Fig. 4.
Protection of Moco activity by recombinant
MocoCP. Free Moco extracted from buttermilk XO and purified
recombinant MocoCP (0.75 µg) were incubated under aerobic conditions.
Samples were taken at the indicated times. Bound Moco was
filtered by spin-column chromatography, and Moco activities were
determined by the Nit1 assay. 100% Moco activity corresponds to 0.4 units/ml.
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ACKNOWLEDGEMENTS |
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We thank M. Macías for technical help and I. Molina and C. Santos for secretarial assistance. We are grateful to Professor R. Mendel, Technical University of Braunschweig, Germany, for the microsequencing of the MocoCP.
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FOOTNOTES |
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* This work was supported in part by Grants PB98-1022-CO-02 and BMC2002-03325 from the Dirección General de Investigación Científica y Técnica, by the Junta de Andalucía (to investigational group PAI CVI-128), and by the Programa Propio de Ayudas a la Investigación of the University of Córdoba.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Max Planck Institute for Plant Breeding Research,
Carl-von-Linne-Weg 10, Cologne 50858, Germany.
§ Present address: Departamento de Bioquímica, Biología Molecular y Genética, Universidad de Extremadura, Cáceres 10071, Spain.
¶ To whom correspondence should be addressed. Tel.: 34-957-218591; Fax: 34-957-218591; E-mail: bb1feree@uco.es.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M211320200
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ABBREVIATIONS |
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The abbreviations used are:
Moco, molybdenum
cofactor;
MocoCP, molybdenum cofactor carrier protein;
NR, nitrate
reductase;
XO, xanthine oxidase;
IPTG, isopropyl-1-thio--D-galactopyranoside;
GST, glutathione S-transferase;
Tricine, N-[2-hydroxy-1,1-bis(hydro-
xymethyl)ethyl]glycine.
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