Coenzymology: biochemistry of vitamin biogenesis and cofactor-containing enzymes
Pantothenate biosynthesis in higher plants
K.M. Coxon*, E. Chakauya*, H.H. Ottenhof*, H.M. Whitney*, T.L. Blundell†, C. Abell‡ and A.G. Smith*1
*Department of Plant Sciences, Downing Street, Cambridge CB2 3EA, U.K., †Department of Biochemistry, 80 Tennis Court Rd, Cambridge CB2 1GA, U.K., and
‡University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, U.K.
Abstract
Pantothenate (vitamin B5 ) is a water-soluble vitamin essential for the synthesis of CoA and ACP (acyl-carrier
protein, cofactors in energy yielding reactions including carbohydrate metabolism and fatty acid synthesis.
Pantothenate is synthesized de novo by plants and micro-organisms; however, animals obtain the vitamin
through their diet. Utilizing our knowledge of the pathway in Escherichia coli, we have discovered and cloned
genes encoding the first and last enzymes of the pathway from Arabidopsis, panB1, panB2 and panC. It
is unlikely that there is a homologue of the E. coli panD gene, therefore plants must make β-alanine by
an alternative route. Possible candidates for the remaining gene, panE, are being investigated. GFP (green
fluorescent protein) fusions of the three identified plant enzymes have been generated and the subcellular
localization of the enzymes studied. Work is now being performed to elucidate expression patterns of the
transcripts and characterize the proteins encoded by these genes.
Introduction
Pantothenate or pantothenic acid is a water-soluble vitamin
more commonly referred to as vitamin B5 . It is synthesized
by micro-organisms and plants; however, it is necessary for
animals to obtain it through their diet [1]. The word pantothenate is derived from the Greek word pantos, meaning
everywhere, due to it being found in most foodstuffs [2].
This fact is supported by a lack of pantothenate deficiencies
reported in nature. Pantothenate is the precursor of the 4 phosphopantetheine moiety of CoA and ACP (acyl-carrier
protein) [3]. CoA and ACP are found in all cell types, with
CoA playing an essential role in central metabolism such
as the tricarboxylic acid cycle, fatty acid oxidation and isoprenoid biosynthesis, and ACP involved in the synthesis of
lipids. In plants CoA is also important in many aspects
of secondary metabolism, including lignin biosynthesis.
From two aspects, the pantothenate pathway is of great
commercial interest. Firstly, the synthesis of pantothenate for
addition to cosmetics, such as hand cream and shampoo,
or to be sold as a vitamin supplement, is carried out on a
large scale, yielding approx. 5000 tons/year [4]. However, this
can be an expensive process due to the necessity of separating
the biologically active D-pantothenate from the L-form in the
racemic mixture. Overexpression of pantothenate in plants
and micro-organisms would not require this purification
step and so, after optimization, may result in more economical
production of the vitamin. Secondly, as pantothenate biosynthesis occurs in plants and micro-organisms and not in
animals, the enzymes of the pathway are ideal targets of
potential herbicides, fungicides and antibiotics.
Key words: Arabidopsis, higher plants, ketopantotate hydroxymethyltransferase (KPHMT),
pantothenate biosynthesis, pantothenate synthetase, subcelluar localization.
Abbreviations used: ACP, acyl-carrier protein; ADC, aspartate decarboxylase; GFP, green
fluorescent protein; α-KIVA, α-ketoisovalerate; KPHMT, ketopantoatehydroxymethyltransferase;
KPR, ketopantoate reductase; PS, pantothenate synthetase.
To whom correspondence should be addressed (email [email protected]).
1
The pantothenate pathway was first elucidated in
Escherichia coli where it is best understood, and known to
consist of four enzymatic steps in two separate branches (see
[26]). The initial step of the pathway involves the transfer of a
hydroxymethyl group from 5,10-methylene tetrahydrofolate
to α-KIVA (α-ketoisovalerate) generating ketopantoate, catalysed by KPHMT (ketopantoate hydroxymethyltransferase).
Ketopantoate is reduced to pantoate by KPR (ketopantoate
reductase) [5] in an NADPH-dependent reaction. β-Alanine
is formed by decarboxylation of L-aspartate catalysed by
L-aspartate-α-decarboxylase in a separate branch of the
pathway [6]. Formation of pantothenate in the final step of
the pathway occurs through an ATP-dependent condensation
reaction between pantoate and β-alanine carried out by PS
(pantothenate synthetase) [7].
Biochemical studies of the pantothenate
pathway in plants
The first indication of the presence of the pantothenate pathway in plants came from the identification of a pantothenaterequiring auxotroph of Datura innoxia (thorn apple) [8,9].
Growth of the mutant tissue culture cells was supported by
addition of ketopantoate, pantoate and pantothenate to the
growth medium. However, addition of α-KIVA had no effect
indicating a deficiency in the capacity to convert α-KIVA to
ketopantoate, in other words absent or defective KPHMT. In
order to determine whether functional KPHMT was present
in the mutant line, Sahi et al. [9] attempted to assay the activity
of the enzyme, but the activity of KPHMT could not be
reliably detected in either the auxotroph or the wild-type.
Further evidence to indicate the presence of the pantothenate pathway in plants, similar to that in bacteria, came
from feeding studies with radiolabelled L-[14 C]valine [10].
Pea (Pisum sativum) leaf discs were fed with radiolabelled
valine from which α-KIVA is derived by transamination.
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HPLC analysis of the different compounds eluted showed
radiolabelled fractions corresponding to α-KIVA, ketopantoyl lactone (derived from ketopantoate) and pantoyl
lactone (derived from either pantoate or pantothenate). The
formation of radiolabelled intermediates along each step of
the pathway known in E. coli suggested the pathway to be
similar in plants, and provided support for the existence of
the enzymes KPHMT and KPR.
Identification of genes encoding
pantothenate biosynthesis enzymes
in plants
Cloning of the genes for pantothenate biosynthesis enzymes
in E. coli has resulted in both sequence information and the
crystal structures of the proteins, which have subsequently
provided valuable tools to search for homologous genes in
higher plants. The identification of these genes has afforded
further insight into the operation of the plant pathway.
KPHMT
KPHMT, the first enzyme of the pathway, is encoded by
panB. The E. coli panB gene was used as a query in a BLAST
search of the Arabidopsis thaliana genome. This resulted in
the identification of two panB genes, panB1 and panB2, with
87% similarity to each other at the amino acid level [11]. The
occurrence of two panB genes in the Arabidopsis genome
is likely to be the result of genome duplication, since the
two genes lie in regions of chromosomes II and III where a
duplication event has taken place [12]. The two panB genes
were amplified from an Arabidopsis cDNA library, and found
to functionally complement the E. coli panB mutant Hfr3000
YA139, indicating that both genes encode active proteins [11].
Furthermore, two panB genes are found in the completed
rice genome, suggesting that the presence of two KPHMT
isoforms might have functional significance in plants.
PS
The final enzyme of the pathway, PS catalyses the ATPdependent condensation of pantoate and β-alanine, generating pantothenate. The E. coli panC mutant was used in
an attempt to identify the panC gene in a number of plant
cDNA libraries. Functional complementation of the auxotroph proved successful when screening the Lotus japonicus
cDNA library [13]. Lotus panC encoded a peptide sequence
of 308 amino acids. An EST (expressed sequence tag) from
rice (Oryza sativum), showing similarity to the E. coli
panC, encoded a 313 amino acid peptide. Neither of the
plant enzymes had an N-terminal extension compared with
the bacterial enzyme, but there was a 20–30 amino acid
insertion in the middle, which corresponds to a region near
the dimer interface of the E. coli PS structure [14]. Overexpression and purification of recombinant Lotus PS allowed
kinetic characterization of the enzyme [15]. Normal saturation kinetics was observed for β-alanine, whereas pantoate
concentrations above 0.5 mM showed inhibition of the activity of the enzyme; the enzyme from E. coli has normal satu
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ration kinetics for both substrates. In contrast, the Lotus
enzyme is not inhibited by its product [15].
After the release of the genome sequence by the Arabidopsis Genome Initiative, the panC gene was identified and was
cloned from an Arabidopsis cDNA library. It was established
to encode a functional protein by complementation of the
E. coli panC mutant.
β-Alanine synthesis
In E. coli, β-alanine is synthesized from L-aspartate via a
decarboxylation reaction catalysed by ADC (aspartate decarboxylase); however, no homologues of ADC were
found in the Arabidopsis genome by BLAST searching.
Furthermore, functional complementation of the E. coli panD
mutant DM 3498 using an Arabidopsis cDNA expression
library resulted in no positive clones. An alternative method
was used to determine whether ADC is encoded in the Arabidopsis genome. FUGUE is a novel search approach, which
compares the query sequence to a database of proteins with
solved crystal structures [15]. Reverse-FUGUE can also be
performed whereby a solved crystal structure can be used as
the query to search a specific predicted proteome. The structures of all the E. coli pantothenate biosynthesis enzymes were
used in a reverse-FUGUE search of the annotated Arabidopsis
proteome. Despite identifying the two genes for KPHMT and
the single gene for PS, no convincing hit for ADC was obtained. Furthermore, no ADC homologue was identified in
Saccharomyces cerevisiae by this approach, indicating that
the gene may not have traversed the prokaryotic–eukaryotic
border [11]. Instead, like other eukaryotes, it is likely that
plants have alternative methods by which β-alanine is produced. In yeast, β-alanine for pantothenate biosynthesis appears to come from the polyamine spermine [16]. The degradation of uracil and thymine to produce CO2 , NH3 , β-alanine
and γ -aminoisobutyrate has been shown to occur in seedlings
of oil seed rape [17] and a gene for one of the enzymes in
maize has been characterized [18]. An alternative method
of β-alanine production, from 3-oxopropanoate, may be
involved in pantothenate production, after the identification
of β-alanine aminotransferase in Arabidopsis [19]. It is still
unclear if any of these methods play a role in the production
of β-alanine specifically for use in the pantothenate pathway.
Furthermore Arabidopsis can survive without the ability to
make spermine [20]. A more detailed explanation of sources
of β-alanine in plants can be found in the review by Raman
and Rathinasabapath [21].
KPR
Owing to the identification of the first and final enzymes of
the pantothenate pathway in the Arabidopsis proteome, it is
inferred that the intermediate enzyme KPR is also present.
Attempts were made to identify KPR by BLAST searches
of the Arabidopsis genome, using the E. coli panE sequence
as the query, but no homologue was found. Since sequence similarity in panE between micro-organisms is poor,
failure to find a homologue of a plant sequence using a
Coenzymology: biochemistry of vitamin biogenesis and cofactor-containing enzymes
Figure 1 Comparison of the enzymes of the pantothenate
biosynthetic pathway from E. coli, Arabidopsis and S. cerevisiae
This figure shows the schematic representation of the alignment of the
mature regions of the enzymes involved in pantothenate biosynthesis,
with the shaded areas showing extensions relative to the E. coli enzyme.
The extensions of the Arabidopsis and S. cerevisiae KPHMT at the
N-termini have been shown to target the enzymes to the mitochondria
[11,24]. The C-termini extensions are of unknown function. The two
eukaryotic PS are known to localize to the cytosol despite the N-terminal
extension seen in the S. cerevisiae enzyme. S. cerevisiae KPR, showing
no extension relative to E. coli, is found in the cytosol [24]. An Arabidopsis
KPR has not yet been identified; however by inference, it is likely to be
found in the cytosol.
microbial sequence might not be surprising. Furthermore,
there is another enzyme, acetohydroxy acid isomeroreductase
(AHIR), encoded by ilvC, which is able to reduce ketopantoate to pantoate [22]. It is unlikely to be involved
in pantothenate biosynthesis in E. coli, since it can only compensate for a panE mutation when it is overexpressed, but
it is possible that in plants it may play a role. Nevertheless,
reverse-FUGUE identified a possible homologue of panE in
Arabidopsis. The protein has a putative nucleotide-binding
motif and the conserved Ser244 thought to be important in the binding of ketopantoate, although the glutamate
associated with catalytic activity of the enzyme was not
conserved [11]. The veracity of this protein being KPR requires further investigation, and will be facilitated by the
generation of an ilvC panE mutant of Salmonella typhimurium [23]. Work is currently underway to test whether the
Arabidopsis gene can rescue the mutant, and to screen plant
cDNA libraries for additional clones that can complement
the auxotrophy.
Subcellular organization of the
pantothenate pathway in plants
Elucidation of the pantothenate pathway in Arabidopsis has
raised many questions. For example, why are there two functional copies of KPHMT in plant proteomes? It is possible
that the proteins are functionally redundant, or it may be that
the two isoforms are localized to discrete parts of the cell.
It is possible that the genes are differentially expressed.
Sequence alignment of KPHMTs from prokaryotes and
eukaryotes clearly shows that the eukaryotic proteins have
N-terminal and C-terminal extensions compared with the
prokaryotic ones (Figure 1), with the possibility that
these are involved in subcellular targeting. To answer this
question, cDNAs encoding C-terminal fusions of GFP (green
fluorescent protein) to the pantothenate biosynthetic enzymes were generated. These constructs were used to transform onion epidermal cells and tobacco epidermal and
guard cells by biolistic transformation. Confocal microscopy
showed both isoforms of KPHMT targeted GFP to the
mitochondria, confirmed by co-localization with formate
dehydrogenase (FDH), an enzyme known to target to the
mitochondria of plants, fused to red fluorescent protein.
Thus both KPHMT1 and KPHMT2 are targeted to the
mitochondria (Figure 2). The discovery that KPHMT is
targeted to the mitochondria led to a breakthrough in the
ability to measure the activity of the enzyme after previous
attempts had proved unsuccessful [13,9]. Purified mitochondria from pea leaves and Arabidopsis suspension cultures
were found to have measurable activity, whereas none was
detected in isolated chloroplasts or boiled mitochondria [11].
In contrast with the mitochondrial location of KPHMT, PSGFP was shown to be cytosolic (Figure 2). This spatial organization of the enzymes implies that either ketopantoate or pantoate must pass out of the mitochondria into the
cytosol. Recent work performed on the subcellular localization of the entire S. cerevisiae proteome has identified both
KPR and PS as cytosolic enzymes, whereas KPHMT is in
the mitochondrion [24]. By inference, the Arabidopsis KPR
homologue may well be cytosolic and thus it is anticipated
that ketopantoate traverses the plant mitochondrial membrane.
It can be concluded that differential subcelluar localization
is not the rationale behind the Arabidopsis proteome containing two functional KPHMTs. It is possible that the two
KPHMT isozymes are differentially expressed. Studies are
being carried out using promoter::GUS fusions, Western-blot
analysis and RT (reverse transcriptase)–PCR to determine the
patterns and levels of expression of the enzymes in higher
plants.
Conclusions
Plants have been shown to possess two functional copies
of the gene, panB, encoding the first enzyme and one copy of
panC, encoding the final enzyme of pantothenate biosynthesis. Possible homologues to KPR have been identified
using structure similarity searchers, but further investigation
is required to determine whether any of these proteins have
KPR activity. Nevertheless, there will be an enzyme that
catalyses this second step of the pathway. In contrast, plants
are unlikely to make β-alanine using ADC. Targeting studies
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Figure 2 Subcellular localization of the Arabidopsis pantothenate biosynthesis enzymes and organization of the pathway in plant cells
Particle bombardment was used to introduce constructs carrying panB1-GFP, panB2-GFP and panC-GFP fusions into onion
epidermal cells. After incubation in the dark for 16–24 h at 25◦ C on 1/2 MS agar, the transformed cells were visualized by
confocal microscopy. The GFP fluorophore was excited using an argon ion laser line of 488 nm and the emission spectra of
the excited GFP were collected between 520 and 580 nm.
using GFP fusions indicated localization of both KPHMT
isoforms to the mitochondria, whereas PS is in the cytosol,
suggesting the need for transportation of either ketopantoate
or pantoate across the mitochondrial membrane.
Once the elucidation of the pathway is complete it may
enable the exploration of its connections to CoA biosynthesis, for which the pathway has been fully elucidated in
plants [25]. In the meantime, we are well situated to start
exploring regulation of the pathway both at the level of gene
expression and fine regulation of enzyme activity. These data
will be invaluable in attempts to overproduce pantothenate
in plants.
We are grateful to EU FPV for funding (HPRN-CT-2002-00244), the
UK Biotechnology and Biological Sciences Research Council (BBSRC)
and the Cambridge Commonwealth Trust.
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Received 28 April 2005