From the Lehrstuhl für Mikrobielle Genetik,
Universität Tübingen, Auf der Morgenstelle 15, Verfügungsgebäude, 72076 Tübingen, Germany, the
¶ Instituto de Biología Molecular y Celular de Plantas,
Universidad Politécnica de Valencia-Consejo Superior de
Investigaciones Científicas, Camino de Vera s/n, 46022 Valencia, Spain, and the
Abteilung für Strukturforschung,
Max Planck Institut für Biochemie,
82152 Planegg-Martinsried, Germany
Received for publication, January 26, 2001, and in revised form, February 26, 2001
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ABSTRACT |
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The Arabidopsis thaliana flavoprotein
AtHAL3a is related to plant growth and salt and osmotic tolerance.
AtHAL3a shows sequence homology to the bacterial flavoproteins EpiD and
Dfp. EpiD, Dfp, and AtHAL3a are members of the homo-oligomeric
flavin-containing Cys decarboxylase (HFCD) protein family. We
demonstrate that AtHAL3a catalyzes the decarboxylation of
(R)-4'-phospho-N-pantothenoylcysteine to
4'-phosphopantetheine. This key step in coenzyme A biosynthesis is
catalyzed in bacteria by the Dfp proteins. Exchange of His-90 of
AtHAL3a for Asn led to complete inactivation of the enzyme. Dfp and
AtHAL3a are characterized by a shortened substrate binding clamp
compared with EpiD. Exchange of the cysteine residue of the conserved
ACGD motif of this binding clamp resulted in loss of
(R)-4'-phospho-N-pantothenoylcysteine
decarboxylase activity. Based on the crystal structures of EpiD H67N
with bound substrate peptide and of AtHAL3a, we present a model for the
binding of (R)-4'-phospho-N-pantothenoylcysteine to AtHAL3a.
Coenzyme A is the principal acyl carrier group in all living cells
and is required for many synthetic and degradative reactions in
intermediary metabolism (1). In bacteria, coenzyme A is synthesized in
five enzymatic steps from pantothenate (2). In the first step,
pantothenate is phosphorylated to 4'-phosphopantothenate by
pantothenate kinase. Then
(R)-4'-phospho-N-pantothenoylcysteine (PPC)1 is
synthesized by the addition of cysteine to 4'-phosphopantothenate. In
the next step, PPC is decarboxylated to
4'-phosphopantetheine (PP). 4'-Phosphopantetheine is
converted to coenzyme A by the enzymes phosphopantetheine
adenylyltransferase and dephospho-CoA kinase.
The coenzyme A biosynthetic pathway is not understood in plants. The
pantothenate kinase has been partially purified and characterized from
spinach (3), but all the other enzymes involved in coenzyme A
biosynthesis in plants are not characterized.
Recently, the flavoprotein AtHAL3a from Arabidopsis thaliana
has been characterized as a protein that is related to plant growth,
salt, and osmotic tolerance (4). AtHAL3a is similar to the flavoprotein
EpiD from Staphylococcus epidermidis, the N-terminal domain
of the Dfp flavoproteins from bacteria (Fig. 1) and to one of the domains of the SIS2
(HAL3) protein from Saccharomyces cerevisiae (4-6). EpiD is
catalyzing the oxidative decarboxylation of peptidylcysteines to
peptidylaminoenthiols and is involved in biosynthesis of the peptide
antibiotic epidermin (7-11), which belongs to the lantibiotics
(12). Dfp was originally described as a flavoprotein involved in DNA
and pantothenate metabolism (13, 14). Recently, it was shown that Dfp
catalyzes the decarboxylation of PPC to PP (6), a
reaction that had been attributed before to a pyruvoyl-containing
enzyme (15). The SIS2 protein influences ion homeostasis and cell cycle
control of S. cerevisiae via the Ppz1p Ser/Thr protein
phosphatase (16-19). Expression of the AtHAL3a gene in
yeast hal3 mutants partially complements their LiCl
sensitivity, and its overexpression in transgenic
Arabidopsis plants improves growth rates and salt and
drought tolerance (4). Environmental salt and drought stress response
in plants include a myriad of cellular and physiological
adaptations ranging from stress signaling mitogen-activated protein
kinase (MAP kinase, MAPK) cascades to Ca2+-mediated sodium
transport and compartmentation by kinase-activated plasma membrane
entry channels and Na+/H+ extrusion and
vacuolar antiports (update reviews are found in Refs. 20-22). The
protective effects observed in AtHAL3a flavoprotein-engineered plants
further confirm the complexity of salt and drought stress tolerance.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Structure-based alignment of A. thaliana AtHAL3a protein with Dfp and EpiD. The
alignment of AtHAL3a, Dfp, and EpiD is taken from Blaesse et
al. (5) and is based on the crystal structures of AtHAL3a (23) and
of EpiD H67N with bound substrate peptide DSYTC (5). The numbering of
the Dfp amino acid residues is based on the experimentally determined
start codon of the dfp gene (6). Residues of EpiD contacting
the substrate peptide DSYTC are in bold letters. Binding of
the substrate peptide to EpiD mainly involves helix H1 of EpiD
(substrate binding helix) and the substrate binding clamp comprising
residues Pro-143 to Met-162. It is assumed that these motifs are also
involved in substrate binding of AtHAL3a and Dfp, respectively.
However, the substrate recognition clamp is shorter by four residues in
AtHAl3a and Dfp compared with EpiD. The His motif contains the strictly
conserved histidine residue of the HFCD proteins (His-90 in case of
AtHAL3a). In front of this His motif, there are sequence insertions in
AtHAL3a and Dfp compared with EpiD. The PXMNXXMW
motif is involved in coenzyme binding and contains a conserved Asn
residue (in bold). In the case of EpiD, this Asn residue was
shown to form a H bond to the carboxylate group of the substrate
peptide.
Molecular characterization of EpiD and the determined enzymatic
function of the Dfp protein indicated that all proteins having a EpiD
homologous domain catalyze the decarboxylation of cysteine residues
(6). This idea was further confirmed by crystal structure analysis of
the active-site mutant EpiD H67N with bound substrate peptide DSYTC.
The substrate of EpiD is embraced by a substrate recognition clamp
comprising residues Pro-143 to Met-162. Substrate binding also involves
an N-terminal substrate binding helix (Fig. 1). The binding clamp and
the substrate peptide form a three-stranded -sheet (5). Residues
such as Asn-117 of the so-called PXMNXXMW motif
of EpiD, which are important for binding of the cysteine residue of the
substrate peptide, are conserved in Dfp and AtHAL3a. EpiD and Dfp are
homododecameric proteins (6), whereas AtHAL3a forms trimers (23). We
therefore proposed that enzymes having a EpiD homologous domain are
homo-oligomeric flavin-containing Cys decarboxylases and suggested the name HFCD
proteins for this protein family (5, 6).
Here, we ascribe an enzymatic function to the flavoprotein AtHAL3a,
demonstrating that AtHAL3a but not the active-site mutant AtHAL3a H90N
catalyzes the decarboxylation of
(R)-4'-phospho-N-pantothenoylcysteine to
4'-phosphopantetheine. The PPC decarboxylases Dfp and
AtHAL3a are distinguished from EpiD by a shortened substrate
recognition clamp containing the sequence motif ACGD (5). In this study we analyze the importance of the conserved cysteine residue of the ACGD
motif for substrate binding of the PPC decarboxylases and
present a theoretical model for the binding of PPC to
AtHAL3a. The data show that 4'-phosphopantetheine and/or
coenzyme A biosynthesis is linked to salt tolerance indicating that
AtHAL3a is not necessarily involved in signal transduction as has been
proposed earlier.
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EXPERIMENTAL PROCEDURES |
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Plasmid Construction
In General-- Polymerase chain reaction amplifications were performed with Vent-DNA-polymerase (New England BioLabs) or PfuTurbo DNA polymerase (Stratagene). The entire sequences of the dfp- and AtHAL3a-coding regions of the constructed plasmids were verified. The used oligonucleotides were purchased from MWG Biotech and Sigma, respectively.
pET28a(+) AtHAL3a H90N-- The mutant AtHAL3a gene was constructed by QuikChange site-directed mutagenesis (Stratagene) using pET28a(+) AtHAL3a (4) as a template. The oligonucleotides (i) forward, 5'-GTGATCCTGTCCTTAACATCGAGCTTAGACGTTG-3', and reverse, (ii) 5'-CAACGTCTAAGCTCGATGTTAAGGACAGGATCAC-3', exchanging the codon CAC (codon 90 of the AtHAL3a-coding sequence) for AAC were used as mutagenesis primers. The pET28a(+)-derived plasmid was transformed into the expression strain Escherichia coli BL21 (DE3) by electroporation. The expression plasmids pET28a(+) AtHAL3a and pET28a(+) AtHAL3a H90N encode N-terminal His tag fusion proteins of the AtHAL3a protein (His-AtHAL3a/His-AtHAL3a H90N: MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGS-AtHAL3a/AtHAL3a H90N).
pQE12 dfp C158A-- The mutant dfp gene was constructed by sequential polymerase chain reaction using pQE12 dfp (6) as a template and cloned into the single EcoRI and BglII sites of the pQE12 expression vector (Qiagen, Hilden, Germany) in such a way that no fusion with the His tag codons occurred. The used terminal primers were (i) forward, 5'-CAATTGTGAGCGGATAACAATTTCAC-3', and (ii) reverse, 5'GGTCATTACTGGATCTATCAACAGG-3'. The oligonucleotides (i) forward, 5'-GGCAGTCAGGCTGCTGGTGATATCGGTCCTG-3', and (ii) reverse, 5'-CAGGACCGATATCACCAGCAGCCTGACTGCC-3', exchanging the codon TGT for GCT were used as mutagenesis primers. The pQE12-derived plasmid was transformed into the expression strain E. coli M15 (pREP4) (Qiagen) by electroporation.
Purification of Proteins
Growth of Strains--
The E. coli strains used were
grown to A578 = 0.4 in 0.5 liters of
B-broth (10 g of casein hydrolysate 140 (Life Technologies, Inc.),
5 g of yeast extract (Difco), 5 g of NaCl, 1 g of
glucose, and 1 g of K2HPO4/liter, pH 7.3)
in 2-liter shaker flasks, induced with 1 mM
isopropyl-1-thio--D-galactopyranoside, and harvested 2 h after induction. E. coli BL21 (DE3) pET28a(+)
AtHAL3a cells were grown in the presence of 100 µg/ml
kanamycin, and E. coli M15 (pREP4) pQE12dfp cells
were grown in the presence of 100 µg/ml ampicillin and 25 µg/ml
kanamycin. The growth temperature was 37 °C.
AtHAL3a and AtHAL3a H90N--
500 ml of
isopropyl-1-thio--D-galactopyranoside-induced E. coli BL21 (DE3) pET28a(+) AtHAL3a/AtHAL3a
H90N cells were harvested and disrupted by sonication in 10 ml of
20 mM Tris-HCl, pH 8.0. 5 ml of the cleared lysate obtained
by two centrifugation steps (each 20 min at 30,000 × g
at 4 °C) was diluted with 5 ml of column buffer (20 mM
Tris-HCl, pH 8.0, 10 mM imidazole, 300 mM NaCl) and applied to an equilibrated Ni-NTA column containing 1 ml of Ni-NTA-agarose (Qiagen). The column was then washed with 10 ml of
column buffer. His-AtHAL3a and mutant His-AtHAL3a H90N proteins, respectively, were eluted with column buffer containing 250 mM instead of 10 mM imidazole, and the yellow
peak fractions (~800 µl) were collected. Immediately after elution
from the column, DTT was added to a final concentration of 5 mM. For gel filtration, a 25-µl aliquot of the Ni-NTA
eluate was subjected to a Superdex 200 PC 3.2/30 column equilibrated in
running buffer (20 mM Tris/HCl, pH 8.0, 200 mM
NaCl) at a flow rate 40 µl/min. The Superdex 200 PC 3.2/30 column and
the standard proteins used for calibration were obtained from Amersham
Pharmacia Biotech. The Ni-NTA and gel filtration eluates were used for
activity assays.
Dfp and Dfp C158A--
500 ml of
isopropyl-1-thio--D-galactopyranoside-induced E. coli M15 (pREP4) pQE-12dfp/pQE-12dfp C158A
cells were harvested and disrupted by sonication in 10 ml of column
buffer (20 mM Tris-HCl, pH 8.0). 5 ml of the cleared lysate
obtained by two centrifugation steps (each 25 min at 30,000 × g at 4 °C) was diluted with 5 ml of column buffer and
loaded on a 1-ml HiTrapQ column (Amersham Pharmacia Biotech)
equilibrated with column buffer. The column was then washed with 5 ml
of column buffer and 5 ml of column buffer containing 0.1 M
NaCl. Dfp was eluted with column buffer containing 0.25 M
NaCl, and the yellow peak fraction (~400 µl) was collected. A
25-µl aliquot of this HiTrapQ eluate was then immediately subjected
to Superdex 200 PC 3.2/30 gel filtration as described above for
AtHAL3a. The fraction containing the maximum amount of Dfp/Dfp C158A
(elution volume 1.02-1.10 ml; Ref. (6)) was used for the activity assay.
SDS-PAGE and Immunoblotting-- Proteins were separated using Tricine-sodium dodecyl sulfate-polyacrylamide (10%) gel electrophoresis (24) under reducing conditions. After SDS-polyacrylamide gel electrophoresis, proteins were electrophoretically transferred to polyvinylidene difluoride membranes (25). AtHAL3a was detected by polyclonal anti-AtHAL3a antiserum. Immuno-reactive proteins were visualized by enhanced chemiluminescence.
Activity Assays and Modeling
AtHAL3a and Dfp Assays-- Approximately 50-100 µg of PPC as Ca2+ salt (6) were incubated with 1-5 µg of Dfp or AtHAL3a for 15-30 min at 37 °C in a total volume of 0.75-1 ml of 50 mM Tris/HCl, pH 8.0, 3 mM dithiothreitol. The mutant proteins AtHAL3a H90N and Dfp C158A, which were supposed to be inactive, were used at approximately double concentration relative to the bound FMN. The reaction mixture was then separated by RPC. The obtained fractions were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS). ESI-FT-ICR-MS was performed using a 4.7-tesla APEXTMII-ESI-FT-ICR mass spectrometer from Bruker Daltonik as described previously (6).
Modeling--
Modeling was done interactively using the
program MAIN (26). The coordinates of the PPC molecule were
generated with the SYBYL molecular modeling software (Version 6.4, Tripos, Inc., St. Louis, MO), which was also used to optimize its
geometry and to minimize the energy of the resulting model.
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RESULTS AND DISCUSSION |
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Purification of His-AtHAL3a H90N--
AtHAL3a and
AtHAL3a H90N were expressed as His-tag fusion proteins and
purified from the corresponding E. coli clones by
immobilized metal affinity chromatography
(IMAC) and additionally by gel filtration (Figs. 2 and
3). Purification of His-AtHAL3a has
already been described (4), but there are only incomplete data for the
molecular weight determination by gel filtration (23). The mutant H90N has the same elution volume as wild-type His-AtHAL3a, and a comparison with standard proteins revealed an apparent molecular mass of 110 kDa
(Fig. 2). The calculated molecular mass of trimeric His-AtHAL3a-FMN is
82 kDa. The ratio of the absorbance values at 280 and 450 nm was not
significantly altered by the H90N mutation. These data show that the
introduced mutation did not affect the trimeric structure and coenzyme
binding of AtHAL3a H90N, indicating that the overall three-dimensional
structure is not significantly altered.
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AtHAL3a Catalyzes the Decarboxylation of
(R)-4'-Phospho-N-Pantothenoylcysteine--
Crystal structure analysis
of flavoprotein EpiD H67N with bound peptide DSYTC showed that
substrate binding of EpiD involves the N-terminal helix H1 of EpiD
(substrate binding helix), the His motif, the
PXMNXXMW motif, and the residues Pro-143 to
Met-162 (substrate recognition clamp) (Fig. 1; Ref. 5). We assume that sequences that are homologous to the substrate binding helix and the
substrate recognition clamp of EpiD are also involved in substrate binding of AtHAL3a and Dfp, respectively. Interestingly, substrate binding helix and substrate recognition clamp of AtHAL3a are more similar to the corresponding sequences of Dfp than that of EpiD. Especially, the substrate recognition clamp of AtHAL3a and Dfp is
shortened by four residues compared with EpiD and contains the
conserved ACGD motif (5). This led to the assumption that AtHAL3 might
be able to decarboxylate
(R)-4'-phospho-N-pantothenoylcysteine as had
already been shown for Dfp, although it had been proposed that AtHAL3a
regulates, via a Ppz1-like protein phosphatase, the expression of genes
related to cell cycle and ion homeostasis (4, 23). To examine this
idea, we incubated His-AtHAL3a purified by IMAC and gel filtration with
PPC. By using RPC separation of the reaction mixture,
it was shown that PPC was converted to 4'-phoshopantetheine (Figs. 4A and
5). The synthesized PPC
contained minor amounts of D-pantothenoylcysteine; however,
ESI-MS data indicated that pantothenoylcysteine was not
decarboxylated by His-AtHAL3a as has also been shown for Dfp
(6).
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At the moment, it is not obvious how the observed activity is correlated with salt and osmotic tolerance mediated by AtHAL3a, since the cofactors 4'-phosphopantetheine and coenzyme A are involved in a lot of different biochemical reactions, and plants use different principles to resist high salt concentrations (reviewed in Refs. 20-22). There is also the possibility that in plants 4'-phosphopantetheine is not only an intermediate in coenzyme A biosynthesis but also in taurine biosynthesis. For bacteria, it is known that taurine has an osmo-protective effect (27). In principle, it is possible that taurine synthesis from PP occurs via cysteamine that is released from PP by a 4'-phosphopantetheinase activity (compare Ref. 28). Plants that overexpressed the AtHAL3a gene showed a faster growth rate than the wild type (4). This effect can be explained by a higher coenzyme A content of AtHAL3a-overexpressing plants.
His90 Is an Active-site Residue of AtHAL3a--
The histidine
residue His-90 of AtHAL3a is conserved in EpiD, Dfp, and all other HFCD
proteins. Recently, it has been shown by site-directed mutagenesis and
crystal structure analysis that this histidine residue is an
active-site residue of EpiD and Dfp (5, 6). For AtHAL3a, a cysteine
residue has been modeled into the active site, and it has been proposed
that oxidation of this cysteine residue occurs via concerted
,
-dehydrogenation and depends on the basic His-90-Glu-77
diad (23).
To verify that His-90 of AtHAL3a is an active-site residue, we investigated the activity of His-AtHAL3a H90N (Fig. 4B). His-AtHAL3a H90N was inactive in decarboxylation of PPC, verifying that the observed activity of AtHAL3a is not due to contaminating Dfp protein of E. coli. A side product of the PPC synthesis was also decarboxylated by AtHAL3a (Fig. 4B) but not by Dfp (not shown), indicating a slightly different substrate specificity of AtHAL3a. This difference in substrate specificity can be related to the different size of the sequence insertion in front of the His motif (see below and Fig. 1).
The Conserved Cys Residue of the Substrate Binding Clamp of
PPC Decarboxylases--
The binding clamp of the peptidylcysteine
decarboxylase EpiD forms a twisted antiparallel -sheet with residues
Ser-152, Ser-153, and Gly-154 in the turn region (5). Ser-153 of EpiD
aligns with the conserved cysteine residue of the ACGD motif of the
PPC decarboxylases (Cys-158 of Dfp and Cys-175 of AtHAL3a).
To investigate the importance of this residue for the
activity/substrate binding of the PPC decarboxylases, we
characterized the Dfp mutant Dfp C158A. Dfp C158A was purified by
anionic exchange chromatography and gel filtration as described for Dfp
(6). Dfp C158A eluted exactly at the same volume as Dfp from the
Superdex 200 PC 3.2/30 column. The ratio of the absorbance values at
280 and 450 nm was not significantly altered by the mutation (not
shown). These data show that the introduced mutation did not affect the
dodecameric structure and coenzyme binding of Dfp C158A, indicating
that the overall three-dimensional structure is not significantly
altered. The mutant protein Dfp C158A was inactive in
decarboxylation of PPC (Fig.
6). Modeling of the enzyme-substrate
complex suggests that Cys-175 of AtHAL3a and Cys-158 of Dfp will be in
direct vicinity of the substrate cysteinyl moiety and might participate
in catalysis (see below and Fig. 7).
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Model for Binding of PPC to AtHAL3a and Reaction Mechanism-- The theoretical model for the binding mode of PPC to AtHAL3a is based on the crystal structure of EpiD with bound peptide DSYTC (Protein Data Bank accession code 1G5Q; Ref. 5) and the known crystal structure of AtHAL3a (Protein Data Bank accession code 1E20 (23)). For modeling, the position of the cysteinyl moiety common to both substrates was taken from the EpiD crystal structure, and an elongated conformation of the PPC molecule was chosen to match the location of the peptide backbone of DSYTC. Both substrates are quite similar in length.
In the resulting model (Fig. 7), the cysteinyl moiety of PPC
is tightly fixed by hydrophobic interactions of its methylene group to
Val-88 and Ile-91. The carboxylate group could form H-bonds to the
backbone NH of Val-88, to the guanidinium group of Arg-95, and to
Asn-142, which is highly conserved within the HFCD family. The presence
of Val-88 constricts the binding site near the FMN considerably
compared with the active site of EpiD and thereby restricts the
rotation of the methylene-thiol moiety. This might be important to
avoid oxidation of the substrate and restrict the overall reaction to a
decarboxylation. The crystal structure of EpiD H67N with bound
substrate peptide DSYTC suggested an FMN-dependent oxidation of S of the cysteine residue, yielding a
thioaldehyde intermediate. It has been assumed that this thioaldehyde
intermediate decarboxylates spontaneously, forming the enethiolate
group of the reaction product (5, 29) and that the decarboxylation of
PPC by AtHAL3a and Dfp follows a similar mechanism. Since we did not find any oxidative decarboxylated product but only
PP, we concluded that the enethiol group is reduced
immediately by FMNH2 of the PPC decarboxylase,
completing the reaction cycle (6).
Favorable hydrophobic interactions between the two methylene groups of
the -alanine part of PPC with Val-30 next to FMN and Leu-173 from the binding clamp and of the dimethyl-methylene group with
Ile-33 and Ile-84 represent a nice match in surface properties. The
phosphate group could be anchored by Lys-34 at the bottom of the
binding site and Lys-171 from the binding clamp. This could explain
how AtHAL3a discriminates between PPC and
pantothenoylcysteine. It is very likely that AtHAL3a does not recognize
peptidylcysteines because the spacing between the peptide bonds will be
different and, therefore, hydrophobic parts in PPC do not
mimic amino acid side chains. The binding clamp crosses the
PPC substrate at the NH-CO-CHOH part of the molecule, with
potential H-bonding contacts to the protein backbone at Leu-173 and
Ala-174. The shorter binding clamp of AtHAL3a places the connecting
loop at Ala-174 to Gly-176 (ACG). The latter residue appears to be
highly conserved within the HFCD family as the corresponding Gly-154
(SSG) in EpiD also marks the turn connecting both
-strands of the
clamp. The position of Cys-175 suggests contacts to the backbone of the
insertion segment, anchoring its position, but after a simple side
chain rotation it could also make a direct contact to a PPC
carboxamide group.
Dfp and AtHAL3a perform the same reaction and, interestingly, show
identical lengths of the substrate binding clamp. The sequence motif
ACGD that presumably forms the turn region within the clamp is present
in both proteins. The difference between AtHAL3a and Dfp is the length
of the insertion in front of the His motif (Fig. 1). Residues Asp-76 to
Leu-89 of AtHAL3a, which connect -strand S3 and
-helix H4,
deviate in the three-dimensional structure from EpiD. An insertion of
eight residues is included and substitutes for the contacts between
trimers present in the dodecameric EpiD (5, 23). Val-88 appears to have
the additional function of constricting the active site of AtHAL3a. Dfp
forms dodecamers and not trimers (6), but an insertion of five residues
is present in Dfp, which might constrict the active site.
Conclusion--
In this paper we have ascribed the PPC
decarboxylase activity to the plant flavoprotein AtHAL3a. This result
is of great importance for the elucidation of the in vivo
role of AtHAL3a in salt and osmotic tolerance. On the other hand, the
presented data are the starting point for a detailed investigation of
coenzyme A biosynthesis in plants. The results confirm that proteins
with an EpiD homologous domain are homo-oligomeric
flavin containing Cys
decarboxylases (HFCD proteins). The elucidation of the
crystal structure of Dfp/AtHAL3a with bound PPC will give
more detailed insights into the substrate binding and the reaction
mechanism of PPC decarboxylases.
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ACKNOWLEDGEMENTS |
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We thank Regine Stemmler and Thomas Dümmler for excellent technical assistance, Michael Uebele for synthesis of (R)-4'-phospho-N-pantothenoylcysteine, and Dietmar Schmid for ESI-FT-ICR-MS experiments.
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FOOTNOTES |
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* This work was supported by the University of Tübingen Strukturfond (to T. K.) and by Research Projects GV-CAPA00-13-C02-02 from Consellería d'Agricultura Generalitat Valenciana and PB98-0565-C04-03 from Ministerio de Educación y Cultura of Spain to P. H-A. and F. A. C-M.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.
§ To whom correspondence and reprint requests should be addressed. E-mail: Thomas.Kupke@t-online.de.
Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.M100776200
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ABBREVIATIONS |
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The abbreviations used are: PPC, (R)-4'-phospho-N-pantothenoylcysteine; PP, 4'-phosphopantetheine; HFCD, homooligomeric flavin-containing Cys decarboxylases; ESI-FT-ICR-MS, electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry; IMAC, immobilized metal affinity chromatography; RPC, reversed phase chromatography; Ni-NTA, nickel nitrilotriacetic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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