From the Department of Biochemistry, St. Jude
Children's Research Hospital, Memphis, Tennessee 38105, the
§ School of Environmental and Evolutionary Biology, Sir
Harold Mitchell Building, University of St. Andrews, St. Andrews, Fife
KY16 9TH, United Kingdom, the ** Department of Microbiology, Monash
University, Clayton, Victoria 3168, Australia, and the
Department of Biochemistry, University of Tennessee,
Memphis, Tennessee 38163
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ABSTRACT |
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Pantothenate kinase (PanK) is the key regulatory
enzyme in the CoA biosynthetic pathway. The PanK gene from
Escherichia coli (coaA) has been previously
cloned and the enzyme biochemically characterized; highly related genes
exist in other prokaryotes. We isolated a PanK cDNA clone from the
eukaryotic fungus Aspergillus nidulans by functional
complementation of a temperature-sensitive E. coli PanK
mutant. The cDNA clone allowed the isolation of the genomic clone
and the characterization of the A. nidulans gene designated
panK. The panK gene is located on chromosome 3 (linkage group III), is interrupted by three small introns, and is
expressed constitutively. The amino acid sequence of A. nidulans PanK (aPanK) predicted a subunit size of 46.9 kDa and
bore little resemblance to its bacterial counterpart, whereas a highly
related protein was detected in the genome of Saccharomyces
cerevisiae. In contrast to E. coli PanK (bPanK),
which is regulated by CoA and to a lesser extent by its thioesters,
aPanK activity was selectively and potently inhibited by acetyl-CoA.
Acetyl-CoA inhibition of aPanK was competitive with respect to ATP.
Thus, the eukaryotic PanK has a distinct primary structure and unique
regulatory properties that clearly distinguish it from its prokaryotic counterpart.
Pantothenate kinase
(PanK)1
(ATP:D-pantothenate 4'-phosphotransferase, EC 2.7.1.33)
catalyzes the first committed step in the universal biosynthetic
pathway leading to CoA. Phosphopantothenate is metabolized rapidly to
CoA (for review, see Ref. 1), which participates as an acyl group
carrier in the tricarboxylic acid cycle, fatty acid metabolism, and
numerous other reactions of intermediary metabolism (2). The
4'-phosphopantetheine portion of CoA is an essential prosthetic group
in a number of enzyme systems including the acyl carrier protein
components of bacterial and eukaryotic fatty acid synthases (3),
citrate lyase (4), ferrichrome synthetase from Aspergillus
quadricinctus (5), and malonate decarboxylase of Malonomonas
rubra (6). 4'-Phosphopantetheine is also required for
E. coli mutants with temperature-sensitive bPanK activity
are also temperature-sensitive for CoA biosynthesis and growth (16). The bPanK gene of E. coli (coaA) was cloned by
functional complementation and found to be identical to a previously
sequenced temperature-sensitive allele called rts (17-19).
E. coli bPanK is a homodimer of 36 kDa subunits which
exhibits highly positive cooperative ATP binding and utilizes a
sequential ordered mechanism with ATP as the leading substrate (20).
CoA and its thioesters inhibit bPanK activity by competitive binding to
the ATP site (20, 21). Nonesterified CoA is the most potent inhibitor
of bPanK in vitro and in vivo, whereas acetyl-CoA
is about 20% as effective as CoA (21). CoA and CoA thioesters also
inhibit mammalian (14, 22, 23) and plant (24) PanK enzymes. Although
both CoA and acetyl-CoA are reported to inhibit these enzymes, in
general, acetyl-CoA is more effective than CoA.
The goal of the present study was to extend the molecular and
biochemical characterization of PanK to eukaryotic cells. Homologs of
E. coli bPanK protein and the coaA gene
(accession no. M90071) are clearly detected in the genomes of
Hemophilus influenzae (accession no. U32746),
Mycobacterium tuberculosis (TIGR gmt7548), Vibrio cholerae (TIGR GVCCS17R), Streptococcus pyogenes
(OUACGT Contig282), and Bacillus subtilis (accession no.
D84432) using standard search and sequence alignment tools. In
contrast, a similar search of the Saccharomyces cerevisiae
genome data base did not reveal the presence of a predicted protein
with significant sequence similarity to the E. coli bPanK
protein or the nucleotide sequence of the coaA gene. Also,
sequences related to bPanK could not be identified in the mammalian
expressed sequence-tagged data base. These results indicated that
eukaryotic cells possess a PanK with a significantly dissimilar primary
structure. Therefore, we employed a genetic selection strategy to clone
a PanK gene from an A. nidulans cDNA library by
functional complementation of the coaA15(Ts) mutant of
E. coli. This paper describes the isolation and
characterization of a eukaryotic PanK with a distinctly different
primary structure and dissimilar regulatory properties compared with
its prokaryotic counterpart.
Materials--
Sources of supplies were: American Radiolabeled
Chemicals, D-[1-14C]pantothenate (specific
activity, 55 mCi/mmol); Appligene, pUC18; Bio-Rad, Bradford dye-binding
protein assay solution; Boehringer Mannheim, Klenow fragment; Analtech
Inc., 250-µm Silica Gel H plates; Bio 101, GeneClean II kit; NEN Life
Science Products, D-[1-14C]pantothenate
(specific activity, 54.5 mCi/mmol) and [ Bacterial Strains and Growth Conditions--
The bacterial
strains used in this work were derivatives of E. coli K-12.
Strain ts9 (leuB6 hisG1 argG6 metB1 rplL9 rts-1 ilu-1 lacY1 gal-6
xyl-7 mtl-2 malA1 tonA2 tsx-1 Isolation of cDNA and Genomic Clones--
An A. nidulans cDNA library was prepared according to the
manufacturer's instructions using the ZAP-cDNA library kit from Stratagene. Messenger RNA (5.1 µg) was isolated from wild-type A. nidulans strain G1071 (completely prototrophic) grown on
A. nidulans minimal medium with 10 mM sodium
nitrate as the sole nitrogen source and in the absence of vitamins (7).
Excision of the
Recombinant A. nidulans phagemid DNA Sequencing--
The genomic DNA (pSTA2000) and cDNA
( Expression Analysis--
Cultures of the wild-type A. nidulans strain GO51 (carrying the biotin auxotrophic marker
bia1) were grown at 30 °C for 16 h in liquid minimal
medium containing 5 mM ammonium tartrate or 10 mM sodium nitrate as sole nitrogen source (27) with or
without a final concentration of 1 µg/ml pantothenate. Total RNA was
extracted from mycelium as described previously (28) and mRNA
prepared using a Quick Prep mRNA purification kit. Northern blot
analysis was carried out as described previously (28) using a 2-kb
BamHI fragment of pSTA2000.
Subcloning of A. nidulans panK cDNA and Expression of
aPanK in E. coli--
The panK cDNA gene from
Aspergillus was amplified from phagemid Construction of bPanK Expression Vector--
The E. coli
coaA gene encoding bPanK was amplified by PCR from pWS7-13-2. A
forward primer (5'-CATATGAGTATAAAAGATCAAACG-3') introduced a
NdeI site at the first translational start and and also
introduced a mutation (E5D) that removed an internal ribosomal binding
site to reduce the occurrence of shorter transcripts. A reverse primer
(5'-GGATCCGAGTATTCGCTCCCCTGCAA-3') added a BamHI site for
subcloning. The PCR was performed using the Advantage cDNA
polymerase mix (CLONTECH), and the product was
ligated into pCR2.1 (Invitrogen). The ligation mixture was transformed
into One Shot cells (Invitrogen). After overnight growth on ampicillin selection medium, plasmid DNA was isolated from a mixture of cells and
digested with NdeI and BamHI. The appropriate
fragment was gel purified and isolated by QIAquick (Qiagen). The
purified DNA fragment was ligated into NdeI- and
BamHI-digested pET-15b (Novagen) treated with calf
intestinal alkaline phosphatase. This ligation mixture was used to
transform strain BL21(DE3) (Novagen), and transformants were screened
for ampicillin resistance and by PCR for the presence of the
coaA insert in the plasmid vector. Single colonies were
isolated and cultured to mid-log phase, frozen at Preparation of aPanK and bPanK Cell Extracts and Purification by
Affinity Chromatography--
Colonies were grown to mid-log phase and
frozen at
The aPanK or the bPanK His-tagged fusion protein was purified in one
step on a Ni-NTA agarose resin (Qiagen) by the same procedure. The
affinity matrix (15 ml) was charged with NiSO4 by
sequentially washing with 60 ml of water, 60 ml of 2 mM
NiSO4, and 90 ml of binding buffer (20 mM
Tris-HCl, pH 7.8, plus 0.5 M NaCl). The activated support
was mixed with the cell extract for 45 min at 4 °C and then packed
into a column. The column was washed successively with 150 ml of the
binding buffer followed by 150 ml of the binding buffer containing 0.04 M imidazole. Bound protein was eluted with 150 ml of
binding buffer containing 0.2 M imidazole. The fractions containing His-tagged aPanK or bPanK were combined and dialyzed against
20 mM Tris-HCl, pH 7.6, 1 mM dithiothreitol,
and 1 mM EDTA and concentrated using an Amicon stirred cell
followed by a Centricon-30 concentrator. The purified proteins were
stored at PanK Assays--
Enzyme preparation and assays were performed as
described previously (21). The PanK specific activities in cell lysates were calculated as a function of protein concentration. Assays were
linear with respect to both time and protein input. Protein concentrations were measured by the method of Bradford (29) with
Isolation of a Eukaryotic PanK by Functional
Complementation--
A cDNA expression library was prepared as
described under "Experimental Procedures" from wild-type A. nidulans, prototrophic strain G1071. The library was transformed
into the E. coli strain ts9, which carried a conditionally
defective rts allele (25) and exhibited poor growth at
37 °C. Selection for growth of the transformants at 37 °C yielded
colonies that harbored library phagemids that functionally complemented
the defect. Three phagemids were purified and retransformed into either
E. coli strains ts9 or DV73. Strain DV73 harbored the
defective coaA15 allele, which expressed a
temperature-sensitive bPanK (16). The coaA and
rts genes are allelic (17). Phagemid designated
The Aspergillus cDNA insert in phagemid Structure and Expression of the panK Gene--
The DNA sequence of
the aPanK cDNA and a 2115-base pair stretch of plasmid pSTA2000
encompassing the panK gene was determined on both strands
(Fig. 1). The panK gene
contained a single open reading frame interrupted by three short
introns typical of those found in fungi (31). The positions of the
introns were confirmed by comparison of the genomic and cDNA
sequences. Although we have not directly determined the amino-terminal
protein sequence for aPanK, the putative translational start is the
first methionine codon of the open reading frame. There was an in-frame
stop codon located 7 codons upstream of the predicted methionine start
codon. The size of the expressed aPanK protein (see below) confirms
this Met as the start site as the next methionine residue is located 154 amino acids downstream. The site of polyadenylation is
approximately 508 nucleotides downstream from the translational stop
codon and is indicated by the arrow in Fig. 1. However, the
precise polyadenylation acceptor could not be determined simply by
comparison of the genomic and cDNA sequences because there is a
stretch of 6 A nucleotides in the genomic sequence at this point. An
AATAAA polyadenylation signal is located 21 nucleotides upstream from
the proposed polyadenylation site.
Northern blot analysis of mRNA prepared from A. nidulans
indicated that the panK gene was transcribed as a single
mRNA (Fig. 2A). The
apparent size of the panK transcript, 1.85 kb, was
consistent with the size predicted from the analysis of the genomic and
cDNA sequences (Fig. 1). The transcript occurred in approximately
the same abundance in cells grown with either ammonium or nitrate as
the nitrogen source, in the presence or absence of pantothenate, and
the level of panK transcript was much lower that that of
A. nidulans actin (data not shown).
Comparison of the Protein Sequences of aPanK, yPanK, and
bPanK--
The predicted protein sequence of aPanK consisted of 420 amino acids with a predicted molecular mass of 46.9 kDa. This sequence was used to perform a similarity search against the S. cerevisiae genome data base. This search identified a predicted
open reading frame (Ydr531w) that consisted of 367 amino acid residues
with a predicted molecular mass of 40.9 kDa. The Ydr531w open reading frame was 44.8% identical and 60.2% similar to the aPanK sequence (Fig. 3). Based on this strong similarity
the Ydr532w open reading frame is predicted to encode the yeast
pantothenate kinase (yPanK). The major difference between aPanK and
yPanK was in the amino-terminal domain, which was significantly longer
in aPanK. We detected only a single PanK isoform in the S. cerevisiae genome.
The similarity between aPanK and the previously described bPanK from
E. coli was far less striking (Fig. 3). bPanK is a protein composed of 316 amino acids with a molecular mass of 36.4 kDa. bPanK
was 16.2% identical and and 32.9% similar to aPanK. These calculations were based on the introduction of several significant gaps
in the bPanK sequence to align the smaller bacterial protein with the
larger aPanK. Nonetheless, the comparison between bPanK and aPanK/yPanK
points to the location of the ATP binding site in aPanK/yPanK. Lysine
101 is a critical residue in bPanK required for the binding of both the
ATP substrate and the CoA regulators to the enzyme (20). The Lys-101
residue in bPanK corresponds to Lys-141 in aPanK and Lys-85 in yPanK as
indicated in Fig. 3, suggesting that these lysine residues may be
involved in nucleotide binding in the aPanK and yPanK proteins.
The Aspergillus panK Gene Encodes a Functional PanK--
The
functional complementation by phagemid Characterization and Kinetic Analysis of aPanK--
The
biochemical properties of aPanK were examined in more detail after the
cloning of the aPanK cDNA into the pET-15b expression vector,
expressing the protein in E. coli strain BL21, and purifying the protein by affinity chromatography as described under
"Experimental Procedures." The aPanK protein was purified in a
single step (Table I), and typically
10-12 mg was obtained from a 500-ml culture of cells (5 × 108/ml) in logarithmic growth. The major variable was the
efficiency of the IPTG induction step before lysis of the cells. The
purified aPanK preparation consisted of a single polypeptide species
(Fig. 2B). The apparent subunit molecular size of the
purified protein, 46 kDa, was consistent with the molecular mass, 46.9 kDa, predicted from the cDNA sequence. The specific activity of the
purified aPanK ranged from 1.5 to 2.5 µmol/min/mg among several
preparations when assayed under the standard conditions described under
"Experimental Procedures" using an ATP concentration of 2.5 mM. The in vitro kinase reaction was optimal
over a broad pH range; and pH values between 6 and 8.5 supported
The affinity constants of the purified aPanK for the two substrates
were determined. The pattern of parallel lines observed in Fig.
4 is characteristic of ping-pong kinetic
systems. However, there are examples of kinases that operate by ordered
bi-bi systems where the lines seem parallel but really are not (32,
33). This effect arises when the kinase has a high affinity for ATP, but because of the relative values of the other rate constants the
apparent Km is much larger. The same thing is
observed when both substrates are varied (Fig. 4). Although the lines
appear parallel, the family of plots intersect far to the left of the (velocity) Regulation of aPanK by CoA and Its Thioesters--
PanK activity
from different sources has been reported to be inhibited by CoA or CoA
thioesters (14, 21-23). We found that acetyl-CoA was the most potent
regulator of the aPanK (see Fig. 6A), and we investigated
the mode of inhibition of enzyme activity (Fig.
5, A and B). The
double reciprocal plot of pantothenate concentration versus
kinase activity indicated that acetyl-CoA inhibited the interaction
between pantothenate and enzyme in a noncompetitive manner (Fig.
5A). In these experiments, acetyl-CoA at 80 µM
reduced pantothenate kinase activity by 66% at 1 mM ATP. In contrast, the interaction of ATP with the enzyme was inhibited in a
competitive manner by acetyl-CoA (Fig. 5B). Acetyl-CoA at 32 µM reduced activity by 51% at 50 µM
pantothenate. The Ki for acetyl-CoA was
calculated as 9 µM from the replot of the slopes from
these data.
The kinetic mechanism of inhibition by a CoA species was the same in
both the pantothenate kinases from Aspergillus and from E. coli (20, 21) in that the inhibitor was competitive with respect to the ATP substrate. However, the pantothenate kinases differed in their sensitivities to either free CoA or acetyl-CoA. We
found previously that bPanK was inhibited potently by nonesterified CoA
(21). Acetyl-CoA and succinyl-CoA inhibited the bacterial enzyme to a
much lesser extent, and malonyl-CoA was without effect (21). Acetyl-CoA
had been reported to inhibit the mammalian pantothenate kinase
activity, whereas free CoA or other CoA thioesters had little or no
effect (14, 22, 23). We investigated the regulation of the purified
aPanK by CoA and its thioesters and directly compared it with
regulation of the bPanK purified from E. coli (Fig.
6, A and B). Both
enzymes were assayed at 1 mM ATP at CoA concentrations up
to 128 µM. Inhibition of aPanK by nonesterified CoA was
not evident, whereas acetyl-CoA was very effective (Fig. 6A). On the other hand, bPanK was inhibited most potently by
nonesterified CoA and acetyl-CoA was considerably less effective,
reducing activity by only 25% at the maximum concentration of
inhibitor (Fig. 6B). Malonyl-CoA was not a potent inhibitor
of either aPanK or bPanK at 1 mM ATP (Fig. 6, A
and B). These data indicated that the ATP binding site of
aPanK had a distinctly different structural context that interacted
selectively with the acetyl moiety of acetyl-CoA as well as the adenine
moiety of either ATP or CoA. The lack of similarity between the primary
structures of aPanK and bPanK, particularly at the predicted nucleotide
binding site (Fig. 3), was consistent with these results.
The identification of the protein sequence for a eukaryotic PanK
will enable the identification and molecular characterization of the
PanK enzymes from higher organisms. The eukaryotic version of PanK
characterized in this report has a primary structure that bears little
resemblance to the previously described enzyme expressed in prokaryotic
cells. The similarities between bPanK and aPanK/yPanK are so weak that
a definitive identification of the eukaryotic PanK in the S. cerevisiae genome is not possible using standard search algorithms
with bPanK as the query. Nonetheless, the eukaryotic aPanK carries out
the same reaction as bPanK, and its ability to complement functionally
the E. coli mutants expressing a temperature-sensitive bPanK
permitted the isolation of the aPanK cDNA. Retrospectively, the
bPanK and the aPanK/yPanK sequences do have some similarities revealed
by the introduction of significant gaps in the bPanK sequence in its
alignment with aPanK (Fig. 3). For example, the critical lysine residue
involved in nucleotide binding in bPanK (Lys-101) (20) appears to be
conserved in aPanK (Lys-141) and yPanK (Lys-85). However, experimental
verification of this point will be essential because the sequences
surrounding Lys-101 in bPanK differ significantly from the sequences
surrounding the "conserved" lysine in aPanK/yPanK. Furthermore, the
sequence, AVDIGGS, between residues 69 and 75 of aPanK, is very similar to a sequence in hexokinase I which is postulated to form part of the
ATP binding pocket (34). By analogy, this sequence motif may also be
part of the ATP site in aPanK, but it is missing from bPanK. The
significance of the other very short regions of identity between these
two proteins remains obscure.
The dissimilarities in the primary sequence of bPanK and aPanK reflect
the distinct differences in the regulatory properties of PanK from
E. coli and A. nidulans. CoA and its thioesters
inhibit bPanK activity by blocking competitively the binding of ATP to the enzyme (20, 21). In contrast, aPanK is regulated selectively by
acetyl-CoA but also in a competitive manner. The advantages afforded by
regulation by different CoA molecular species may relate to the
differences in CoA subcellular compartmentation and metabolism.
E. coli is capable of synthesizing pantothenate (1) and in
fact, produces and secretes 15 times more pantothenate than it requires
for CoA biosynthesis (13). Acetyl-CoA is an end product of glucose
metabolism and is highest in E. coli growing on glucose as a
carbon source (21). Thus, the decreased sensitivity of bPanK to
acetyl-CoA allows this pool to expand and accommodate the metabolic
demands of rapidly dividing cells. In contrast, mitochondria and
peroxisomes are considered to have the highest concentration of CoA and
its thioesters in eukaryotes (35-39). The PanK enzyme, which is
rate-limiting also in mammalian CoA biosynthesis (14), is cytosolic
(14, 22, 23). Thus, PanK is not located in the correct cellular
compartment to sense a significant proportion of the total CoA pool
directly. Its high degree of sensitivity to acetyl-CoA points to a
mechanism for low concentrations of this specific CoA species to
control the cellular levels of total CoA by modulating activity of this
key regulatory enzyme.
The selectivity of aPanK for acetyl-CoA suggests that the ATP binding
site is distinctly different from that found in bPanK or other
ATP-utilizing enzymes. The amino acids flanking the lysine in the
proposed ATP binding site in aPanK (Fig. 3) do not resemble either a
type A or a type B nucleotide binding site (40) and may represent a
unique ATP binding fold. A comparison of the crystal structure of bPanK
with that of aPanK may reveal the residues that determine the
specificity for the acetyl moiety as well as enhance our understanding
of the divergent structures which catalyze identical enzymatic reactions.
INTRODUCTION
Top
Abstract
Introduction
References
-(L-
-aminoadipyl)-L-cysteinyl-D-valine synthetase, the first enzyme for penicillin biosynthesis in fungi including Aspergillus nidulans (7). Escherichia
coli is capable of de novo pantothenate biosynthesis,
and a sodium-dependent permease actively transports
pantothenate into the cell in both bacteria (8-10) and mammals (11,
12). However, metabolic labeling experiments in E. coli (13)
and rat heart (14, 15) show that the utilization, rather than the
supply, of pantothenate controls the rate of CoA biosynthesis. In fact,
E. coli produces 15-fold more pantothenate than is required
for maintaining the intracellular CoA level (13). This excess
pantothenate is excreted into the medium.
EXPERIMENTAL PROCEDURES
-32P]-dCTP
(specific activity, 3000 Ci/mmol); Fisher Scientific, Scintisafe 30%;
Amersham Pharmacia Biotech, Quick-Prep mRNA purification kit;
Promega, restriction endonucleases and T4 DNA ligase; Qiagen, P100
columns; Schleicher & Schuell, nitrocellulose; Sigma, CoA, acetyl-CoA
and malonyl-CoA; Stratagene, ZAP-cDNA synthesis kit; Whatman, DE81
filter circles. All other materials were reagent grade or better.
R
supE44) (25) was obtained from the Coli
Genetic Stock Center, Yale University. Strain ts9 was conditionally
defective and did not grow at 42 °C. Complementation of the
temperature-sensitive rts-1 allele by the coaA
gene (pWS7-13-2) encoding E. coli PanK permitted
phosphopantothenate and CoA biosynthesis (17, 18). Strain ts9 also
exhibited a temperature-sensitive growth requirement for isoleucine
because of the defect in the ilv-1 allele. Strain DV73
(coaA15 srl::Tn10 recA metB1
relA1 spoT1 gyrA216
R
F
) was unable to synthesize phosphopantothenate and CoA
at 42 °C because of a specific defect in the coaA15
allele that encoded a temperature-sensitive (bPanK) protein (16). Rich
medium was Luria broth (26), and minimal medium consisted of medium E
salts (26) supplemented with glucose (0.4%), thiamine (0.001%), and required amino acids (0.01%). Agar (1.5%) was added to media
formulations for plating. The ampicillin concentration was 50 µg/ml
when indicated.
-ZAP yielded 2.5 × 108 phagemids
in 30 µg of DNA. E. coli strain ts9 was electroporated (2,500 volts, capacitance 25 microfarads, pulse time 4.88 ms) with 0.5 µg of the excised cDNA library and incubated at 23 °C for
2 h. Transformants were plated onto Luria broth agar plus ampicillin at 37 °C. Plasmid pWS7-13-2 encoding the E. coli bPanK was the transformation and phenotypic positive control.
STA1999 was digested
with KpnI and SpeI, and the 450-base pair
fragment was separated from the 1.5-kb and 2.9-kb vector sequences by
gel electrophoresis in 1% agarose. The fragment was purified using a
GeneClean II kit and then used as template for preparation of a
radiolabeled probe together with [32P]dCTP, dATP, dTTP,
dGTP, and Klenow fragment. Southern blots prepared from A. nidulans chromosomes I-VIII (Fungal Genetic Stock Center) were
hybridized with the probe. Blots were washed at 25 °C in 2 × saline sodium citrate and exposed overnight at
70 °C. Hybridizing
cosmids were purified, digested with EcoRV, EcoRV plus XhoI, or BamHI plus BglII, and
transferred to Zeta-Probe membrane for rescreening. The membrane was
then hybridized with the KpnI/SpeI fragment of
phagemid
STA1999 and washed at 65 °C in 1 × saline sodium
citrate. One of the four positive cosmids, W19B06 (34C5), was purified
using a Qiagen P100 column and digested with a number of restriction
enzymes. Genomic DNA fragments were separated by agarose gel
electrophoresis, blotted onto Zeta-Probe membrane, and probed with the
KpnI/SpeI fragment derived from phagemid
STA1999. The probe hybridized with a 5.0/5.1-kb doublet band from an
XbaI digest of the cosmid. The two genomic DNA fragments were separated, purified, and subcloned into pUC18 that had been digested with XbaI. Hybridization of the two genomic
DNA fragments with the A. nidulans phagemid
STA1999 cDNA fragment identified the 5.1-kb clone (plasmid
pSTA2000) as containing the genomic DNA corresponding to the gene
designated panK.
STA1999) sequences were determined on both strands by automated DNA
sequencing using an Applied Biosystems 373A automated fluorescent
sequencing apparatus and a PRISM Ready Reaction dideoxy terminator
cycle sequencing kit (Applied Biosystems) with primers at a
concentration of 10 µM and 0.5 µg of pSTA2000 as
template. Sequences were assembled using Sequencher (Gene Codes Corp.).
The cDNA sequence of panK was verified independently by
automated DNA sequencing at the Molecular Resource Center of St. Jude
Children's Research Hospital.
STA1999,
encoding aPanK. The forward primer created a novel restriction site for
NdeI at the amino-terminal methionine and removed an
internal BamHI site (5'-GTCATATGTCCGCCACTGATCCTACTC-3'). The
reverse primer introduced a BamHI site downstream of the
stop codon (5'-AGGATCCGGTTGCCGCCTAAGCTCAT-3'). A polymerase chain
reaction (PCR) was performed using Advantage cDNA polymerase mix
(CLONTECH), and the product was ligated into the TA
cloning vector pCR2.1 (Invitrogen). The ligation mixture was
transformed into E. coli One Shot cells (Invitrogen). After
overnight growth, plasmid was isolated from the ampicillin-resistant
population of cells and digested with NdeI and
BamHI, and the appropriate fragment was gel purified by
QIAquick (Qiagen). The purified fragment was ligated into
NdeI and BamHI digested pET-15b (Novagen) treated
with alkaline calf intestinal phosphatase. This ligation mixture was
used to transform E. coli strain BL21(DE3) (Novagen), and
ampicillin-resistant transformants were screened for the correct
insertion by PCR.
70 °C in the
presence of 7% dimethyl sulfoxide, and screened for overexpression of
bPanK protein by SDS-polyacrylamide gel electrophoresis after IPTG
induction. Plasmids were recovered from several clonal cultures that
overexpressed the His-tagged protein of the appropriate molecular size.
These plasmids were each transformed into strain DH5
for subsequent
plasmid purification and DNA sequencing to verify the amino acid
sequence of the bPanK protein encoded by the plasmid.
70 °C in the presence of 7% dimethyl sulfoxide and
tested for their ability to overexpress a protein of the appropriate
size after IPTG induction. Overnight cultures were prepared as a series
of 1:100 dilutions made directly from the thawed freeze down; the
following morning, cultures in early-mid log phase were added to 500 ml
of LB and grown to a density of approximately 5 × 108
cells/ml. IPTG was then added to a final concentration of 1 mM, and incubation was continued for a further 3 h at
37 °C. Cells were collected by centrifugation in a JA-10 rotor
(8,000 rpm, 4 °C, 10 min) and stored at
20 °C overnight. The
cells were lysed by the addition of 1 mg/ml DNase and lysozyme plus
0.1% Triton X-100. The cells were then frozen at
70 °C for 2 h, thawed on ice, and the soluble extract was isolated by
centrifugation in a Ti 45 rotor at 40,000 rpm for 3 h.
20 °C in the presence of 50% glycerol. The purity of
the protein preparations was assessed by SDS-gel electrophoresis on
12% polyacrylamide gels.
-globulin as a standard. Standard assays contained
D-[1-14C]pantothenate (45.5 µM;
specific activity 55 mCi/mmol), ATP (2.5 mM, pH 7.0),
MgCl2 (2.5 mM), Tris-HCl (0.1 M, pH
7.5), and 32 µg of protein from a 35-60% ammonium sulfate fraction
in a total volume of 40 µl. The mixture was incubated for 10 min at
37 °C, and the reaction was stopped by depositing a 30-µl aliquot
onto a Whatmann DE81 ion exchange filter disc that was washed in three changes of 1% acetic acid in 95% ethanol (25 ml/disc) to remove unreacted pantothenate. 4'-Phosphopantothenate was quantitated by
counting the dried disk in 3 ml of scintillation solution.
RESULTS
STA1999
complemented the temperature-sensitive growth defect in both bacterial
mutants on rich medium, albeit yielding slightly smaller colony
diameters than the control colonies arising from transformation with
the positive control plasmid, pWS7-13-2, expressing the E. coli
coaA gene. Limited growth of strain DV73 (coaA15(Ts))
on rich medium at 42 °C is often observed because of a large
preexisting CoA pool coupled with the high level of amino acid
supplementation (16, 30). Therefore, transformation of strain DV73 with
phagemid
STA1999 was repeated, and ampicillin-resistant colonies
were selected at the permissive temperature, 30 °C. Subsequently, 48 colonies were scored for the temperature-dependent growth
phenotype on glucose-minimal medium at 42 °C. All 48 colonies grew
at the nonpermissive temperature, verifying that complementation was not caused by reversion of the host strain phenotype. These data clearly indicated that the A. nidulans cDNA expressed
from phagemid
STA1999 encoded the functional equivalent of an active
pantothenate kinase.
STA1999 was
used to screen a bank of genomic clones representing A. nidulans chromosomes I-VIII obtained from the Fungal Genetic
Stock Center as described under "Experimental Procedures." Genomic
DNA was blotted onto membranes and hybridized with a
32P-labeled probe derived from the 450-base pair
KpnI/SpeI fragment of phagemid
STA1999.
Positive cosmids from the first screen were identified as W19B06,
W21A12, W21H08, W23E02, W23D11, W24H12, and W24H03 from chromosome III.
Cosmid W19B06 (34C5) was digested with a panel of restriction enzymes,
and the fragments were separated by agarose gel electrophoresis and
blotted onto membranes. Hybridization with the 450-base pair
STA1999
probe signaled a region of the gel containing a 5.0/5.1-kb doublet from
an XbaI digest. Purification of the two DNA bands and
reprobing identified the 5.1-kb fragment as containing the genomic
sequences for the cDNA insertion in phagemid
STA1999. The gene
for A. nidulans pantothenate kinase (aPanK) was designated
panK, and the genomic fragment containing this gene was
subcloned to yield plasmid pSTA2000.
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Fig. 1.
DNA sequence and predicted amino acid
sequence of the panK. The numbers on
the right refer to nucleotides relative to the A of the
start codon (number +1), and the numbers on the
left refer to amino acid residues of the predicted aPanK
coding sequence. Introns are shown in lowercase, and the
arrow indicates the approximate site of polyadenylation.
Isolation and DNA sequence analysis of the cDNA and genomic clones
used to generate this figure are described under "Experimental
Procedures."
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Fig. 2.
Expression of the panK gene
and analysis of the purified aPanK protein. Panel A,
analysis of mRNA expression from the panK gene by
Northern blot. mRNA was isolated from A. nidulans cells
grown in A. nidulans minimal medium (7) with 5 mM ammonium tartrate as the sole source of nitrogen and
containing 1 µg/ml pantothenate. RNA was hybridized with a
32P-labeled probe prepared from the 2-kb BamHI
fragment of pSTA2000. Molecular size markers (in kb) are indicated on
the left. Panel B, analysis of the purified aPanK
His-Tag fusion protein by SDS-gel electrophoresis. The His-Tag aPanK
fusion protein was expressed from plasmid pET15b/aPanK and was purified
by metal ion chelate affinity chromatography from E. coli
cell extracts as described under "Experimental Procedures."
Molecular mass markers (in kDa) are indicated on the
right.
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Fig. 3.
Alignment of the predicted amino acid
sequences of aPanK, bPanK, and yPanK. The predicted amino acid
sequence of aPanK determined in this study was compared with the
predicted sequence of the Ydr531w gene product (accession no. 927798)
identified in the S. cerevisiae genome by a sequence
similarity search using the aPanK sequence. aPanK is also compared with
E. coli bPanK (the product of the coaA gene).
Highlighted residues are those that are identical between
the aPanK protein and either yPanK or bPanK. The lysine residue
highlighted with white on black indicates the location of
Lys-101, the ATP binding site on bPanK (20) and its alignment with
Lys-141 in aPanK and Lys-85 in yPanK. In the percent similarity
calculations, similar amino acid groups were defined as follows: P, A,
G, S, T; Q, N, E, D; H, K, R; C; V, L, I, M; and F, Y, W.
STA1999 coupled with the
cDNA/genomic DNA sequence analysis strongly indicated that the
Apergillus panK gene encoded a functional PanK. This point
was tested by assaying extracts from strain DV73
(coaA15(Ts)) transformed with phagemid
STA1999 for PanK
activity. Because strain DV73 had a temperature-sensitive bPanK (16),
extracts from strain DV73 transformed with the empty control plasmid
possessed a low background PanK specific activity. Transformation of
strain DV73 with phagemid
STA1999 resulted in a dramatic increase in PanK activity in the soluble fractions of the cells (data not shown).
These results were consistent with the complementation studies and
demonstrated that the protein expressed by phagemid
STA1999 was a
functional PanK.
80% of the maximum activity (data not shown). Cofactors were not
required for the reaction, and the addition of Zn2+ was
inhibitory at
50 µM. Of the other cations tested,
the addition of Ca2+, Co2+, or Mn2+
did not affect activity in the standard assay mixture. Other nucleotides could replace ATP somewhat in the assay, with GTP supporting up to 72% of the reaction (Table
II). Manganese could replace
Mg2+ up to 48% (Table
III).
Purification of aPanK
Nucleotide Specificity of aPanK
Cation Specificity of aPanK
1 axis and far below the (substrate
concentration)
1 axis. The apparent Km
for pantothenate at saturating ATP concentrations was 60 µM (Fig. 4A), and the Km
for ATP at saturating pantothenate concentrations was 145 µM (Fig. 4B).
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Fig. 4.
Kinetic characterization of aPanK.
Homogeneous aPanK was assayed for activity with various concentrations
of pantothenate (4-62.5 µM) in the presence of 20 ( ),
80 (
), or 320 (
) µM ATP (panel A). aPanK
was also assayed for activity with various concentrations of ATP
(20-320 µM) in the presence of 16 (
), 32 (
), or 64 (
) µM pantothenate (panel B). Double
reciprocal plots of the initial velocities indicate a
Km of 60 µM for pantothenate and a
Km of 145 µM for ATP at saturating
concentrations of the second substrate.
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Fig. 5.
Kinetic analysis of inhibition by
acetyl-CoA. Homogeneous aPanK was assayed for activity with
various concentrations of pantothenate (4-62.5 µM)
without inhibitor ( ) or in the presence of 20 (
), 40 (
), or 80 (
) µM acetyl-CoA (panel A). aPanK was also
assayed for activity with various concentrations of ATP (20-320
µM) without inhibitor (
) or in the presence of 8 (
), 16 (
), or 32 (
) µM acetyl-CoA (panel
B). Double reciprocal plots indicated that inhibition of aPanK by
acetyl-CoA was noncompetitive with respect to pantothenate and
competitive with respect to ATP.
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Fig. 6.
Inhibition of aPanK or bPanK activities by
acetyl-CoA or CoA. Homogeneous preparations of aPanK (panel
A) or bPanK (panel B) were assayed for activity at 1 mM ATP and 45.5 µM (1 µCi of
D-[1-14C]pantothenate) with various
concentrations (8-128 µM) of free CoA, acetyl-CoA, or
malonyl-CoA as indicated. The specific activity of aPanK was 2.33 µmol/min/mg and of bPanK was 2.25 µmol/min/mg in these assays.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Dr. A. J. Clutterbuck, University of Glasgow, for sending us A. nidulans strains. We also thank Dr. H.-W. Park, Department of Structural Biology, St. Jude Children's Research Hospital, for helpful discussion.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM 45737 (to S. J.) and GM 34496 (to C. O. R.), the European Commission through Biotech Contract ERB BIO2CT942100 (to J. R. K.), the Monash Research Fund (to S. E. U.), Cancer Center (CORE) Support Grant CA 21765, and the American and Lebanese Syrian Associated Charities.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF098669.
¶ Present address: MRC Laboratory for Molecular Cell Biology, University College London, Gower St., London, WC1E 6BT, U. K.
Recipient of a Wellcome Trust travel grant for carrying out
part of this work in Australia.
§§ To whom correspondence should be addressed: Dept. of Biochemistry, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Tel.: 901-495-3494; Fax: 901-525-8025; E-mail: suzanne.jackowski{at}stjude.org.
The abbreviations used are:
PanK, pantothenate kinase; aPanK, A. nidulans pantothenate kinase; yPanK, Saccharomyces cerevisiae pantothenate kinase; bPanK, Escherichia coli (bacterial) pantothenate kinase; kb, kilobases; PCR, polymerase chain reaction; IPTG, isopropyl-thio--D-galactopyranoside.
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REFERENCES |
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