From the Department of Applied Biotechnology, DuPont Pharmaceuticals Company, Wilmington, Delaware 19880-0336
Received for publication, October 26, 2000, and in revised form, January 4, 2001
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ABSTRACT |
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Pantothenic acid and Pantothenic acid (vitamin B5) is a metabolic precursor
to coenzyme A (CoA) and acyl carrier protein, which are cofactors
required by a large number of metabolic enzymes. Biosynthesis of
pantothenic acid occurs in microbes and plants only, whereas animals
obtain it in their diet. In bacteria, it is synthesized by the
condensation of pantoate, derived from 2-oxoisovalerate, an
intermediate in valine biosynthesis, and -alanine are metabolic
intermediates in coenzyme A biosynthesis. Using a functional screen in
the yeast Saccharomyces cerevisiae, a putative amine
oxidase, encoded by FMS1, was found to be rate-limiting for
-alanine and pantothenic acid biosynthesis. Overexpression of
FMS1 caused excess pantothenic acid to be excreted into the
medium, whereas deletion mutants required
-alanine or pantothenic
acid for growth. Furthermore, yeast genes ECM31 and
YIL145c, which both have structural homology to
genes of the bacterial pantothenic acid pathway, were also required for
pantothenic acid biosynthesis. The homology of FMS1 to
FAD-containing amine oxidases and its role in
-alanine biosynthesis suggested that its substrates are polyamines. Indeed, we found that all
the enzymes of the polyamine pathway in yeast are necessary for
-alanine biosynthesis; spe1
, spe2
, spe3
, and spe4
are all
-alanine
auxotrophs. Thus, contrary to previous reports, yeast is naturally
capable of pantothenic acid biosynthesis, and the
-alanine is
derived from methionine via a pathway involving spermine. These
findings should facilitate the identification of further enzymes and
biochemical pathways involved in polyamine degradation and pantothenic
acid biosynthesis in S. cerevisiae and raise questions
about these pathways in other organisms.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-alanine, produced by the
decarboxylation of L-aspartate (1, 2). In Escherichia
coli, four genes, panB, panC,
panD, and panE, encode the four enzymes
required for pantothenic acid biosynthesis, as illustrated in Fig.
1 (3).
View larger version (13K):
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Fig. 1.
The pantothenic acid pathway in E. coli. The diagram shows the relationship of the
E. coli genes and the putative relationships of the yeast
genes to the pantothenic acid pathway. ECM31 and
panB encode ketopantoate hydroxymethyltransferase (EC
2.1.2.11), YHR063C and panE encode
2-dehydropantoate 2-reductase (EC 1.1.1.169), and YIL145C
and panC encode pantoate- -alanine ligase (EC 6.3.2.1).
The yeast genome lacks a panD homolog encoding aspartate
1-decarboxylase (EC 4.1.1.11).ilvGCD, (EC
4.1.3.18, EC 1.1.1.86, EC 4.2.1.9).
Previous work has indicated that the yeast Saccharomyces
cerevisiae requires exogenous pantothenic acid for growth (4). This growth requirement can be replaced by -alanine (5). This implies that yeast have all the enzymes required for pantothenic acid
biosynthesis except for aspartate-1-decarboxylase, the enzyme necessary
for
-alanine biosynthesis in E. coli (1, 2). Consistent
with this, a structural homolog of aspartate-1-decarboxylase is absent
from the proteome of yeast (6), whereas structural homologs of all the
other enzymes of the pantothenic acid pathway do exist in yeast. The
gene ECM31, thought to be involved in cell wall maintenance
(7), has homology to panB of E. coli and
Aspergillus nidulans (8). The gene YIL145c is a
panC ortholog, encoding pantothenate synthase, and has been
shown to be functional in E. coli (9). The putative
YHR063c gene has structural homology to
panE, as noted in the Yeast Proteome Data base (6). Thus, the specific absence of a gene for aspartate-1-decarboxylase may appear
to be consistent with the observation, first reported almost 60 years
ago, that yeast require exogenous pantothenic acid for growth (4).
Decarboxylation of aspartate is not the only pathway for -alanine
biosynthesis. In some E. coli mutants, the source of
-alanine for pantothenic acid biosynthesis involves reduction of
uracil to dihydrouracil followed by hydrolysis first to
-ureidoproprionate and second to CO2, NH3,
and
-alanine (10). In addition, degradation of polyamines by amine
oxidases can produce
-alanine (11, 12), effectively making
-alanine from methionine (13). However, polyamine metabolism has
never been implicated previously in pantothenic acid biosynthesis. We
were therefore interested in the putative amine oxidase encoded by the
yeast gene FMS1, which was originally identified as a
multicopy suppressor of fen2 pantothenic acid import mutants
and encodes a protein of 508 amino acids with sequence homology to
FAD-containing amine oxidases (14). Pantothenic acid uptake deficiency
in fen2 mutants causes CoA limitation, which affects yeast
growth primarily by limiting ergosterol biosynthesis, suggesting a
related role for FMS1 (5, 14, 15).
In this report we show that S. cerevisiae can synthesize
-alanine and is therefore capable of de novo biosynthesis
of pantothenic acid. Furthermore, the biochemical pathway of
-alanine synthesis differs from that found in bacteria. We have
found that
-alanine is formed from spermine via the amine oxidase
encoded by FMS1. Thus, the
-alanine moiety of pantothenic
acid is derived from methionine via S-adenosylmethionine and
the polyamine pathway. These findings should facilitate the elucidation
of other enzymes and metabolic intermediates involved in polyamine
degradation and pantothenic acid biosynthesis and raise questions about
these metabolic pathways in other organisms.
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EXPERIMENTAL PROCEDURES |
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Yeast Strains and Media--
The parental yeast strains BY4741
and BY4742 and their gene deletion derivatives (16) from the
Saccharomyces Deletion Project were obtained through
Research Genetics (Huntsville, AL); these strains are as
follows: BY4741 (MATa his3 leu2 met15 ura3); BY4742 (MAT his3 leu2 lys2 ura3); BY4741-0595 and
BY4742-10595 (fms1
); BY4741-5757 and BY4742-15757
(fen2
); BY4741-3316 and BY4742-13316 (ecm31
); BY4741-2304 and BY4742-12304
(YIL145c
); BY4741-5034 and BY4742-15034
(spe1
); BY4741-1743 and BY4742-11743
(spe2
); BY4741-5488 and BY4742-15488
(spe3
); BY4741-6945 and BY4742-16945 (spe4
). JHT14 (MAT
abz1
::HIS3 his3 trp1 ura3) was made
during this study. The abz1
::HIS3
allele was constructed by micro-homologous recombination (17) with a
DNA construct made in a single step by the polymerase chain
reaction, using oligonucleotide polymerase chain reaction
primers (Sigma-Genosys, The Woodlands, Texas) designed as
previously described for the trp
mutants (18). Growth and manipulation of yeast strains (19, 20) was on "YNB-P" medium, which
was mixed from the individual chemicals. YNB-P had the same recipe as
"Yeast Nitrogen Base with ammonium sulfate" (Difco, Detroit, MI),
except that pantothenic acid was omitted. Amino acid supplements (18)
and 2% glucose were also present in YNB-P medium. Pantothenic acid and
other supplements were added to YNB-P as indicated in "Results" and
the figure legends. YPD medium was 2% glucose, 2% bactopeptone, and
1% yeast extract (Difco). Pantothenic acid,
-alanine, spermine,
spermidine, and putrescine (Sigma) were prepared as stock solutions in
water and used in media at the concentrations indicated in the
figure legends.
Multicopy Suppressor Screen--
Yeast strain JHT14 was
transformed with a yeast high copy library (21) and ~5 × 104 Ura+ transformants were pooled, divided
into aliquots, and stored in 25% glycerol at 70 °C. Transformants
were then spread at a density of 105 Ura+ cells
per 10-cm Petri dish on synthetic agar medium containing 2% glucose,
but lacking uracil, adenine, histidine, methionine, and pantothenic
acid. After incubation for 3 days at 30 °C, rapidly growing colonies
occurred at a frequency of ~1.5 × 10
4. The rapid growth phenotype of 37 of 55 colonies tested was found to be plasmid-dependent, based on
the lack of growth on a selective medium containing 5-fluoroorotic acid
and uracil (22). Ten of these plasmids were recovered from yeast by
transformation of E. coli and confirmed to confer the rapid
growth phenotype on selective medium when reintroduced into yeast
strain JHT14. Based on comparison with sequence data in the
Saccharomyces Genome Data base, six plasmids contained the
ABZ1 locus, and four plasmids contained the FMS1
locus. The ABZ1 plasmids enhanced growth only because of the
low concentration of paraaminobenzoic acid used in the medium, and they
were not studied further.
Plasmids and DNA Manipulations--
E. coli strains
DH5 and DH10B (Life Technologies, Inc.) were used for DNA
manipulations by standard methods (23). Plasmids were introduced into
yeast using lithium acetate (24). Yeast DNA isolation for recovery of
plasmids in E. coli was carried out using the Yeast Plasmid
Isolation kit (Bio 101, Carlsbad, CA). The FMS1 coding
sequence was amplified by polymerase chain reaction using
oligonucleotide primers Fms1For-Xho (5'-ccctcgagatgaatacagtttcaccag-3') and Fms1Rev-Bam (5'-ttggatccctatttcagtaagtcag-3') and ligated into the
SalI and BamHI sites of YEp195AC (25) to create
the ADH1-FMS1 overexpression vector. The E39Q
substitution was made using QuikChange (Stratagene, La Jolla,
CA) and mutagenic primers FmsE39Qupper (5'-gtcttgttcttcaggccagagatc-3')
and FmsE39Qlower (5'-gatctctggcctgaagaacaagac-3'). The DNA sequence of
the entire mutant open reading frame was confirmed subsequently.
"Cross-feeding" Experiments--
Log phase cultures
of strain BY4742-10595 (fms1) or strain BY4742-13316
(ecm31
) containing vector YEp195AC were prepared in
synthetic medium lacking uracil and washed by centrifugation in water,
and ~105 cells were spread on 10-cm Petri dishes
containing synthetic agar medium lacking uracil and pantothenic acid.
Log phase cultures of strain BY4742 and fen2
,
fms1
, and ecm31
deletion derivatives harboring
either YEp195AC or the ADH1-FMS1
overexpression plasmid were prepared in synthetic medium lacking
uracil, washed by centrifugation in water, and spotted onto the
ecm31
and fms1
"lawns" at a
density of ~107 cells per 5 µl. Plates were incubated
at 30 °C for 2 days, after which time "halos" of growing lawn
cells formed around spots of cells that excreted pantothenic acid or
downstream metabolites into the medium.
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RESULTS |
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FMS1 Overexpression Enhances Growth in the Absence of Pantothenic
Acid--
A high copy yeast genomic DNA library was screened for genes
that enhanced growth in the absence of pantothenic acid, and plasmids
containing genomic DNA in the region of the FMS1 locus were
found. To determine whether FMS1, rather than other DNA
sequences in these plasmids, was responsible for the enhanced growth,
the FMS1 open reading frame was subcloned into an expression
vector under the control of the ADH1 promoter and confirmed
to have a DNA sequence identical to the published sequence (Ref. 14,
GenBankTM accession number X81848). This plasmid was
introduced into yeast, and it was found that ADH1-FMS1, but
not the empty vector, could enhance the growth of yeast on medium
lacking pantothenic acid (Fig. 2).
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FMS1, ECM31, and YIL145c Are Required for Pantothenic Acid
Production in Yeast--
As shown above, yeast grew well in the
absence of pantothenic acid when FMS1 was overexpressed.
This finding prompted us to test the currently available deletion
strains, fms1, ecm31
, YIL145c
, and fen2
for pantothenic acid and
-alanine auxotrophy (see Fig. 1). The deletion strains and parental
strain BY4742 were plated on medium lacking pantothenic acid and
-alanine or on medium supplemented with these compounds (Fig.
3A). The fms1
strain required either pantothenic acid or
-alanine for growth. This
is consistent with the results for overexpression of FMS1; overexpression of FMS1 enhanced growth in the absence of
pantothenic acid/
-alanine, whereas the fms1
deletion
totally blocked growth in the absence of pantothenic
acid/
-alanine.
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In the same experiment, the ecm31 and
YIL145c
strains could utilize pantothenic acid but
differed from the fms1
strain because they could not grow
on
-alanine. Neither the fen2
nor the parental strain
required these supplements. Other potential metabolites, including
-ureidoproprionate, 5,6-dihydrouracil, l-aspartic acid, and
1,3-diaminopropane (100 µM) did not support growth of the
deletion strains (data not shown). The same results were also obtained
using a different parental strain, BY4741, and its deletion derivatives
(data not shown). Thus, based on the auxotrophic phenotypes,
FMS1 is required for
-alanine production, whereas
ECM31 and YIL145c are required downstream in the
pantothenic acid pathway (Fig. 1).
Further evidence that FMS1 functions in the same pathway as
ECM31 was obtained from a complementation analysis using the
ADH1-FMS1 overexpression plasmid. This plasmid was
introduced into fms1, ecm31
, and
fen2
strains, which were then tested for growth in the
absence of
-alanine (Fig. 3B). Growth occurred in the
fms1
and fen2
strains, but not the
ecm31
strain, indicating that FMS1 is
dependent on ECM31 for pantothenic acid biosynthesis. Thus,
S. cerevisiae does not require exogenous pantothenic acid or
-alanine in the medium for growth, and FMS1 activity is
rate-limiting for
-alanine biosynthesis under the conditions used.
Overexpression of FMS1 Results in Excretion of Excess
Metabolites--
Dramatically increased metabolic activity in the
pantothenic acid pathway caused by FMS1 overexpression can
be detected in cross-feeding experiments, in which cells
excreting excess pantothenic acid cause halos of growth in lawns
of pantothenic acid auxotrophs. Dense spots of wild-type,
fen2, fms1
, and ecm31
strains
containing either the ADH1-FMS1 overexpression vector or the
empty vector were placed on lawns of either ecm31
or
fms1
cells on medium lacking pantothenic acid (Fig.
4). After incubation, halos formed around
each of the FMS1-overexpressing strains, with the exception of the ecm31
strain, on both lawns. No halos formed
around strains harboring empty vector. The simplest explanation for the
halos is that FMS1 overexpression results in excretion of
excess pantothenic acid, which is then taken up by the lawn cells,
allowing them to grow. Overexpression of FMS1 in the
ecm31
strain did not result in a halo, further confirming
that FMS1 and ECM31 function in the same
pathway.
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The FAD-binding Domain of Fms1p Is Necessary for -Alanine
Production--
Although it is required for
-alanine production,
FMS1 encodes a protein that has no structural homology to
bacterial aspartate-1-decarboxylases. Instead, Fms1p has homology to
FAD-containing amine oxidases (14) and likewise contains a
GXGXXG dinucleotide-binding motif similar, for
example, to Candida albicans Cbp1p, human
monoamine oxidases A and B, and the peroxisomal acetylspermidine
oxidase Aso1p of Candida boidinii (Fig.
5A). To assess the role of FAD
in
-alanine production, we made the E39Q substitution mutant,
equivalent to the substitution that was shown to abolish FAD binding
and catalytic activity of monoamine oxidase B (26-28). The resulting
ADH1-fms1(E39Q) expression plasmid was introduced into the
fms1
strain, and transformants were tested for growth in
the absence of
-alanine and pantothenic acid (Fig. 5B).
The E39Q mutant did not complement the
-alanine and pantothenic acid
auxotrophy of the fms1
strain, consistent with a role for
FAD in the mechanism of
-alanine production.
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FMS1 Links Polyamine Biosynthesis with Pantothenic Acid Production
in Yeast--
The sequence homology of Fms1p to amine oxidases and the
apparent role of FAD in -alanine production by Fms1p suggested that polyamines could provide the substrates for Fms1p. We therefore tested
deletion mutants of the polyamine pathway (29, 30) for
-alanine
auxotrophy. Parental, spe1
, spe2
,
spe3
, spe4
, fms1
, and
ecm31
strains were plated on medium lacking pantothenic acid,
-alanine, and polyamines or on medium supplemented with pantothenic acid,
-alanine, spermine, spermidine, or putrescine (Fig. 6). All four of the
spe
mutants were able to grow when one of the compounds
spermine,
-alanine, or pantothenic acid was added to the medium. In
addition, spe1
and spe3
could also grow on
spermidine, and spe1
could grow on putrescine. As
expected, the fms1
and ecm31
strains could
grow on pantothenic acid but could not utilize any of the polyamine
compounds. Thus, biosynthesis of
-alanine and pantothenic acid is
dependent on the polyamine biosynthetic pathway, consistent with
production of
-alanine via polyamine degradation (11-13). The
source of the carbon atoms in
-alanine would therefore be from
methionine via spermine. Based on these results, the relationship of
the polyamine pathway to pantothenic acid biosynthesis and the key
genes involved are illustrated in Fig. 7.
The spe
strains were not able to grow on the potential
polyamine degradation metabolite 1,3-diaminopropane (data not shown),
apparently ruling out this compound as an intermediate in
-alanine
biosynthesis.
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DISCUSSION |
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S. cerevisiae Can Synthesize -Alanine and Pantothenic
Acid--
Yeast have been reported to require a supplement of either
pantothenic acid or
-alanine, from which it has been inferred that
they cannot synthesize pantothenic acid de novo (4, 5). Contrary to this, we found that overexpression of the yeast gene FMS1, encoding a putative amine oxidase, allowed strong
growth on medium lacking pantothenic acid. Furthermore, when
FMS1 was overexpressed under the control of the
ADH1 promoter, excess pantothenic acid was excreted from the
cells. Thus, yeast clearly have the capacity to synthesize pantothenic
acid de novo when FMS1 is overexpressed. To
eliminate the possibility that the pantothenic acid biosynthesis was a
metabolic abnormality caused by FMS1 overexpression, we analyzed gene deletion mutants. The fms1
strains were
auxotrophic for
-alanine and could grow when supplemented with
either
-alanine or pantothenic acid. Deletions in genes that have
structural homology to the bacterial genes of the pantothenic acid
pathway, ECM31 and YIL145c (see Fig. 1), caused
pantothenic acid auxotrophy, but these strains did not grow on a
-alanine supplement, indicating that these genes are downstream in
the pathway (see Fig. 1). These auxotrophic phenotypes indicate that
FMS1, ECM31, and YIL145c are normally
involved in pantothenic acid biosynthesis. Direct evidence that
FMS1 and ECM31 are in the same pathway came from the finding that FMS1 requires ECM31 activity to
make pantothenic acid; the ecm31
deletion eliminated both
the ability of FMS1 to enhance growth in the absence of
pantothenic acid and also eliminated its ability to cause pantothenic
acid excretion. Thus, pantothenic acid biosynthesis is a natural part
of metabolism in S. cerevisiae, and production of the
-alanine required involves a putative amine oxidase, encoded by
FMS1.
Spermine Is Required for -Alanine Biosynthesis in
Yeast--
Three different enzymatic pathways have been shown to
produce
-alanine: decarboxylation of aspartate (1, 2), degradation of pyrimidines (10), and degradation of polyamines (13). The polyamine
pathway has not been implicated previously in pantothenic acid
biosynthesis, and spermine, in particular, has no previously identified
physiological function in yeast (31). The involvement of the polyamine
pathway is suggested by the Fms1p amino acid sequence, which has
structural homology to FAD-containing amine oxidases (14), some of
which are involved in the oxidative degradation of polyamines (4). In
addition, we showed that a FAD binding site mutant of FMS1,
E39Q, did not complement the fms1
mutant, consistent with
a role for oxidation by the Fms1p protein. We therefore investigated
deletion mutants of the polyamine pathway to see whether this pathway
is required for
-alanine synthesis. Indeed, spe1
,
spe2
, spe3
, and spe4
mutants
were all auxotrophic for
-alanine on a medium that lacked
polyamines. This showed that synthesis of spermine is required for
-alanine biosynthesis in yeast. A more detailed analysis of which
polyamine pathway intermediates could support growth of the
spe
and fms1
mutants confirmed this
conclusion (Fig. 6). Thus, in yeast,
-alanine is derived from
methionine via spermine, making polyamine degradation part of
pantothenic acid biosynthesis (Fig. 7).
We found that the auxotrophic phenotypes of the spe
mutants for polyamines were readily observable on minimal synthetic
medium in the absence of
-alanine or pantothenic acid (Fig.
6). This contrasts with previous reports, in which special
precautions in medium preparation were required, such as HCl washing of
glassware and avoidance of autoclave use, to eliminate contaminating
amines, and in which many cell divisions were required to deplete
intracellular pools of polyamines (32). The difference is simply in the
presence or absence of
-alanine or pantothenic acid; in their
absence, a relatively high level of polyamine metabolism is required to meet
-alanine requirements, such that contaminated glassware and
intracellular pools do not make a significant contribution. In the
presence of
-alanine or pantothenic acid, as customary in yeast
media, low levels of contaminating polyamines are sufficient for
essential processes, such as hypusine synthesis (33), which are
unrelated to the pantothenic acid pathway.
The Pathway from Spermine to -Alanine--
Amine oxidases have
been shown to catalyze a number of different degradation reactions for
polyamines, producing various aldehydes and amines, such as
3-aminopropanal and 1,3-diaminopropane, respectively (13). Thus, the
simplest hypothesis is that the Fms1p enzyme converts spermine to
3-aminopropanal and spermidine and that aldehyde dehydrogenases, for
which there are seven genes in yeast (34), would be required to convert
the 3-aminopropanal to
-alanine. A less direct route between
spermine and
-alanine could, in principle, involve the intermediate
1,3-diaminopropane (13). However, this compound was not able to support
growth of the spe
mutants in the absence of
-alanine
and therefore appears not to be on the pathway in yeast. The simple
phenotype of
-alanine auxotrophy in yeast will help identify the
metabolic intermediates and additional enzymes involved.
Regulation of FMS1 Activity--
It may seem unexpected that yeast
would have the capacity to make pantothenic acid and yet require a
supplement for efficient growth on customary yeast media. In
fact, on medium containing glycerol or acetate as the sole carbon
source, we found that pantothenic acid and -alanine were not
rate-limiting for growth (data not shown). Thus, FMS1
activity is growth-limiting only on glucose medium. This simple
observation may explain the carbon source-dependent phenotype (catabolite repression) reported for the fen2
pantothenate transporter mutant (15). In the light of the finding that
pantothenic acid biosynthesis is a natural part of yeast metabolism, we
propose that growth of the fen2 mutant, which cannot absorb
pantothenic acid from the medium, depends on pantothenic acid synthesis
inside the yeast cells. The fen2 mutant would therefore be
growth-limited on glucose because of insufficient FMS1
expression caused by the presence of glucose. Likewise, wild-type
strains would depend on internal synthesis when pantothenic acid was
absent from the medium and would be growth-limited by insufficient
FMS1 expression on glucose medium. These observations
suggest that FMS1 activity is regulated and raise questions
concerning the mechanism of regulation of the pantothenic acid pathway
in yeast.
-Alanine Biosynthesis in Other Organisms--
The finding that
-alanine biosynthesis is different between yeast and bacteria raises
questions as to how other organisms, such as fungi and plants, make
-alanine. At the present time in the public sequence data bases
there are over a dozen identifiable aspartate-1-decarboxylase genes
from different prokaryotic species, whereas this enzyme does not appear
to be present in eukaryotic species. In contrast, proteins of
significant sequence homology to Fms1p can be found in eukaryotes, in
particular in plants, but not in prokaryotes. The closest sequence
similarity to Fms1p is in Cbp1p from the yeast C. albicans,
a protein with steroid binding activity (14, 35). This suggests that
plants and lower eukaryotes generally produce
-alanine and hence CoA
by a polyamine degradation pathway, as described here for yeast.
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ACKNOWLEDGEMENTS |
---|
We thank our colleagues at DuPont Pharmaceuticals: Greg Hollis, Karyn O'Neil, and Shaoxian Sun for their critical evaluation and help in writing the manuscript and Julie Bunville, Karen Krakowski, and Laura Bolling for DNA sequencing.
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FOOTNOTES |
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* 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 should be addressed: DuPont Pharmaceuticals
Company, Experimental Station E336/239A, Rte. 141 and Henry Clay Rd.,
Wilmington, DE 19880-0336. Tel.: 302-695-9831; Fax: 302-695-9420;
E-mail: jeremy.h.toyn@dupontpharma.com.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M009804200
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