A novel isoform of pantothenate synthetase in
the Archaea
Silvia Ronconi, Rafal Jonczyk and Ulrich Genschel
Lehrstuhl fu
¨
r Genetik, Technische Universita
¨
tMu
¨
nchen, Freising, Germany
Pantothenate is the essential precursor to CoA, which
is of central importance for all parts of metabolism.
This is shown by the fact that more than 400 enzyme-
catalyzed reactions are known to involve CoA (KEGG
database [1]). Many more enzymes utilize acylated
forms of CoA or require the CoA-derived phospho-
pantetheine as a prosthetic group. Typically, plants,
fungi and microorganisms are able to synthesize panto-
thenate de novo, whereas animals rely on pantothenate
in their diet.
Pantothenate synthetase (PS) catalyzes the last step in
the biosynthesis of pantothenic acid, also known as vita-
min B
5
. The enzyme (EC 6.3.2.1) has been extensively
studied in Escherichia coli [2,3], Mycobacterium tubercu-
losis [4,5], and Arabidopsis thaliana [6], and is highly
conserved in the Bacteria and Eukaryota. Bacterial PS
(Eqn 1) generates pantothenate from pantoate and
b-alanine. It is an AMP-forming synthetase that
proceeds via an acyl-adenylate intermediate and belongs

fied two conserved archaeal protein families as the best candidates for the
missing steps. Here we report the characterization of the predicted PS gene
from Methanosarcina mazei, which encodes a hypothetical protein
(MM2281) with no obvious homologs outside its own family. When
expressed in Escherichia coli, MM2281 partially complemented an auxo-
trophic mutant without PS activity. Purified recombinant MM2281 showed
no PS activity on its own, but the enzyme enabled substantial synthesis of
[
14
C]4¢-phosphopantothenate from [
14
C]b-alanine, pantoate and ATP when
coupled with E. coli pantothenate kinase. ADP, but not AMP, was
detected as a coproduct of the coupled reaction. MM2281 also transferred
the
14
C-label from [
14
C]b-alanine to pantothenate in the presence of panto-
ate and ADP, presumably through isotope exchange. No exchange took
place when pantoate was removed or ADP replaced with AMP. Our results
indicate that MM2281 represents a novel type of PS that forms ADP and
is strongly inhibited by its product pantothenate. These properties differ
substantially from those of bacterial PS, and may explain why PS genes, in
contrast to other pantothenate biosynthetic genes, were not exchanged
horizontally between the Bacteria and Archaea.
Abbreviations
COG, clusters of orthologous groups; KPHMT, ketopantoate hydroxymethyltransferase; KPR, ketopantoate reductase; PANK, pantothenate
kinase; PS, pantothenate synthetase.
2754 FEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBS

steps in archaeal CoA biosynthesis [10]. (COG protein
family identifiers cited in this report are defined in the
COG database [15]).
Here we report the characterization of the predicted
PS gene from Methanosarcina mazei. We conclude that
the COG1701 family represents the archaeal isoform
of PS, which utilizes the same substrates as bacterial
PS, but forms ADP instead of AMP and has distinct
kinetic properties. This supports the view that the
intermediates of pantothenate biosynthesis are univer-
sally conserved, whereas the corresponding enzymes
were recruited independently in the Bacteria and
Archaea.
Results
Prediction of conserved archaeal protein families
for PS and PANK
Genomic context and phylogenetic pattern analysis
previously identified the COG1701 and COG1829 pro-
tein families as the best candidates for the missing
steps leading from pantoate to 4¢-phosphopantothenate
in archaeal CoA biosynthesis [10]. Meanwhile, many
more archaeal genomes have been completed, and the
comparative genomics search for the missing steps was
repeated by using the STRING tool [16]. This analysis
revealed additional, previously undetected, links
between established archaeal CoA genes and the
COG1701 and COG1829 families, confirming that the
latter are strong candidates for archaeal PS and
PANK (Fig. 1).
Fig. 1. The CoA biosynthetic pathway and its reconstruction in the

ei; Fig. 2). A straightforward general function predic-
tion is possible for the COG1829 family, which
belongs to a superfamily of small molecule kinases
(GHMP kinases [17]) and is therefore proposed to rep-
resent archaeal PANK. This leaves COG1701, an
orphan family with no obvious links to other protein
families, as the best candidate for archaeal PS. Using
the hhpred prediction server [18], COG1701 was
found to be a distant homolog of acetohydroxyacid
synthase, which ligates two molecules of pyruvate to
yield acetolactate. Specifically, there is approximately
20% sequence identity between COG1701 proteins and
the b-domain of acetohydroxyacid synthases. This
domain has no specific catalytic function but is
thought to be important for the structural integrity of
acetohydroxyacid synthase [19].
Functional complementation of an E. coli
panC mutant
In the genome of Me. mazei, the predicted ORF for
PS (MM2281) is situated in a potential operon
together with the predicted PANK gene and the dfp
gene (Fig. 2), and this cluster is therefore expected to
cover the CoA biosynthetic steps leading from panto-
ate to 4¢-phosphopantetheine. The MM2281 ORF was
cloned by PCR and tested for its ability to comple-
ment the E. coli panC mutant strain AT1371 in liquid
minimal medium (Fig. 3). The panC gene, which
encodes PS in E. coli, and the empty pBluescript KS
vector served as positive and negative controls in this
experiment, respectively. All transformants grew well

M pantothenate (not shown).
Pantothenate synthetase from Methanosarcina mazei S. Ronconi et al.
2756 FEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBS
medium. This might be caused by an endogenous non-
specific activity able to produce pantothenate or by the
emergence of revertants. Nevertheless, regardless of the
actual reason for this behavior, the observation that
MM2281-carrying cells recovered more quickly from
pantothenate starvation and grew faster than the nega-
tive control in two independent experiments indicates
that expression of MM2281 partially complements the
auxotrophic phenotype of E. coli AT1371.
PS activity of recombinant MM2281
The MM2281 protein was overproduced as an N-ter-
minal His-tag fusion protein in E. coli and had a sub-
unit molecular mass in good agreement with its
predicted size (30 kDa as judged by SDS ⁄ PAGE). The
native molecular mass of MM2281 estimated by gel
filtration was 57 000 Da, indicating that the enzyme
is apparently a dimer in solution.
Purified recombinant MM2281 was checked for its
ability to synthesize pantothenate from pantoate,
b-alanine and ATP by using a sensitive isotopic assay
procedure. However, we were not able to demonstrate
PS activity of MM2281 alone. Even after incubation
for 3 h, the amount of [
14
C]b-alanine converted into
[
14

14
C]b-alanine into [
14
C]pantothenate (Fig. 4, lanes 2
and 3). E. coli PANK alone did not act on [
14
C]b-ala-
nine but was conducive to quantitative formation of
[
14
C]4¢-phosphopantothenate when coupled with E. coli
PS (Fig. 4, lanes 4 and 5). As the products of E. coli
PS and E. coli PANK are firmly established, the reac-
tions with these enzymes provide chromatography
standards for pantothenate and 4¢-phosphopantothe-
nate and also confirm that the E. coli PANK prepara-
tion used here was not contaminated with detectable
PS activity. Therefore, the [
14
C]4¢-phosphopanto-
Fig. 4. Synthesis of pantothenate or 4¢-phosphopantothenate
through MM2281 (Me. mazei PS) or helper enzymes. Standard
enzyme assays were carried out as described in Experimental
procedures, containing no enzyme (control), individual enzymes,
or enzyme combinations, as indicated. The figure shows the
14
C-labeled products after a reaction time of 3 h. Separation was
achieved by TLC. The enzyme abbreviations are as follows: EcPS,
E. coli PS; EcPANK, E. coli PANK; PyrK, rabbit pyruvate kinase;
bAla, b-alanine; PA, pantothenate; PPA, 4¢-phosphopantothenate.

a
ND
E. coli PANK ND ND
E. coli PS + E. coli PANK ND > 900
a
MM2281 + E. coli PANK ND 94 ± 11
b
MM2281 + PyrK ND ND
MM2281 + PyrK +
E. coli PANK
ND 140 ± 22
b
a
With respect to E. coli PS. This value is a lower estimate because
the reaction was complete within the first interval.
b
With respect
to MM2281.
S. Ronconi et al. Pantothenate synthetase from Methanosarcina mazei
FEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBS 2757
thenate produced by the combined action of MM2281
and E. coli PANK clearly demonstrates the capacity of
MM2281 to synthesize pantothenate from pantoate
and b-alanine (Fig. 4, lane 6). The rate of 4¢-phospho-
pantothenate synthesis in this assay corresponds to a
PS activity of 94 nmolÆmin
)1
Æmg
)1
with respect to

Combining the above constants gives the overall equi-
librium constant for Eqn (1) at approximately pH 8
and 25 °C(K¢
Eqn (1)
= 7.2 · 10
7
). The large value
means that the reaction in Eqn (1) will go to comple-
tion under physiological conditions, including the
enzyme assay used in this study (pH 8.0, 37 °C).
Given the large effect of removing pantothenate on
MM2281, we also tested the effect of removing the
possible coproduct ADP. ADP was removed by pyru-
vate kinase, which generates ATP from ADP in the
presence of excess phosphoenolpyruvate. This system
did not detectably accelerate pantothenate synthesis by
MM2281 alone but, interestingly, increased the rate of
4¢-phosphopantothenate formation through MM2281
and E. coli PANK approximately 1.5-fold (Fig. 4,
lanes 7 and 8).
With a view to directly observing possible adenosine
nucleotide coproducts of MM2281-catalyzed pantothe-
nate synthesis, standard assays were analyzed by using
a TLC system that provides separation of ATP, ADP,
and AMP (Fig. 5). Whereas MM2281 alone had no
discernible hydrolytic activity towards ATP, the cou-
pled reaction of MM2281 and E. coli PANK generated
a substantial amount of ADP. AMP was not detected
as a coproduct, suggesting that MM2281 is not an
AMP-forming PS according to Eqn (1). By compari-

2758 FEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBS
K¢
Eqn (2)
= 3.9 · 10
5
. Although K¢
Eqn (2)
is smaller
than K¢
Eqn (1)
, the reaction shown in Eqn (2) will still
essentially go to completion in our enzyme assay or
under physiological conditions.
MM2281-catalyzed pantothenate–b-alanine
isotope exchange
The role of ATP and ADP in the MM2281-catalyzed
de novo synthesis of pantothenate could not be investi-
gated independently, because the assay for this forward
activity required the presence of E. coli PANK, which
utilizes ATP and generates ADP. In order to circum-
vent this problem, we assayed MM2281 alone for its
ability to catalyze an isotope exchange between [
14
C]b-
alanine and pantothenate (Table 2). The cosubstrate
dependence of this exchange activity then allowed con-
clusions about the role of adenosine nucleotides and
the mechanism of MM2281. Generally, isotope
exchange between a given substrate–product pair
occurs in the presence of all cosubstrates and coprod-

i
) was removed from the
Eqn (2) system in addition to ATP, there was no sig-
nificant further reduction in exchange activity, showing
that both ATP and P
i
are dispensable. However, when
both ATP and ADP were removed, the resulting
exchange activity was negligible. In summary,
MM2281 catalyzed significant transfer of
14
C-label
from b-alanine to pantothenate in presence of pantoate
and ADP. When pantoate was removed, the resulting
pantothenate–b-alanine exchange was negligible. Also,
the exchange reaction occurred only in the presence of
adenosine nucleotide, and ADP but not AMP could
satisfy this requirement.
Again, this behavior shows that the MM2281 prepa-
rations were not contaminated with E. coli PS, because
bacterial PS requires only AMP to catalyze the panto-
thenate–b-alanine isotope exchange [4,6]. The data in
Table 2 also show that, apart from pantoate, both
ATP and ADP have a strong effect on the rate of the
MM2281-catalyzed pantothenate–b-alanine exchange
reaction. The simplest explanation for this behavior is
that pantoate, ATP and ADP are all substrates or
products of MM2281, which is consistent with the
notion that the enzyme is a synthetase that drives
pantothenate formation by hydrolysis of ATP.

ues indicate that the cosubstrate combination indicated was not
tested. ND, not detectable.
Reactants
Initial exchange rate
a
(%)
Experiment I Experiment II
Eqn (2)
Complete system 100 100
Minus pantoate 2
Minus ATP 46 50
Minus ATP, minus pantoate 3
Minus ATP, minus P
i
45
Minus ATP, minus ADP 3
Eqn (1)
Complete system 20
Minus pantoate 1
Minus ATP ND
Minus ATP, minus pantoate ND
a
Normalized to the value in the complete system of Eqn (2), which
was equal to 2.5 and 1.5 · 10
)3
Æmin
)1
in Experiments I and II,
respectively.
S. Ronconi et al. Pantothenate synthetase from Methanosarcina mazei

this reaction clearly favors hydrolysis of pantothenate.
Thus, based on Eqn (3) and K¢
Eqn (3)
, the majority of
the pantothenate in the isotope exchange assay used
here would be converted to pantoate and b-alanine.
Using K¢
Eqn (3)
and the initial concentrations of panto-
ate, b-alanine and pantothenate in the assay, the
maximum fraction of
14
C-label associated with panto-
thenate at equilibrium would be 35%. However, we
observed that [
14
C]pantothenate accumulated up to
60% of the total
14
C-label during the assay. This cor-
responds to an equilibrium constant of ‡ 1 ⁄ 15, which
is much larger than the reported value for K¢
Eqn (3)
.
Discussion
Experimental confirmation of computationally
predicted archaeal PS
Metabolic reconstruction of the CoA biosynthetic
pathway in representative organisms previously
revealed that the Archaea lack known genes for the

strong support for the prediction that COG1701 repre-
sents the archaeal PS protein family.
Properties of MM2281 (Me. mazei PS)
Our data suggest that MM2281 is an ADP-forming
pantothenate synthetase (Eqn 2) that is subject to
strong product inhibition by pantothenate. The behav-
ior of MM2281 in the isotope exchange experiments
clearly suggests that MM2281 is an adenosine nucleo-
tide-dependent pantothenate synthetase and not a
reversible pantothenate hydrolase. On the basis of the
large values of the equilibrium constants for Eqns (1,2)
(see above), the equilibria of both reactions can be
assumed to lie on the side of pantothenate formation.
In other words, regardless of the type of synthetase
reaction, coupling of pantothenate synthesis from pan-
toate and b-alanine to the hydrolysis of ATP will drive
the equilibrium to the product side. Therefore, the
strong acceleration of MM2281-catalyzed pantothenate
synthesis by the removal of pantothenate is very prob-
ably not due to a shift in the equilibrium of the reac-
tion, leaving potent inhibition of MM2281 by
pantothenate as the best explanation. We propose that
MM2281 is an ADP-forming synthetase according to
Eqn (2), because this is consistent with the observation
that the synthesis of 4¢-phosphopantothenate through
MM2281 and E. coli PANK was accompanied by the
accumulation of ADP but not AMP (Fig. 5) and accel-
erated by removing ADP. Furthermore, the isotope
Pantothenate synthetase from Methanosarcina mazei S. Ronconi et al.
2760 FEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBS

(MM2282; Fig. 2), which may be more effective than
E. coli PANK in accelerating MM2281. Moreover,
conserved phylogenetic profiles and chromosomal
proximity indicate a strong functional link between
archaeal PS (COG1701) and archaeal PANK
(COG1829) (Fig. 1). Also, lack of an interacting pro-
tein required for optimal activity could explain why
expression of MM2281 achieved only partial comple-
mentation of the E. coli panC mutant (Fig. 3). How-
ever, our attempts to express and purify MM2282 did
not meet with success (data not shown), so this
hypothesis could not be tested.
The observation that MM2281 facilitated the panto-
thenate–b-alanine exchange in the absence of ATP and
P
i
may be taken to indicate that MM2281 is a Ping
Pong enzyme able to catalyze a partial reaction. How-
ever, the isotope exchange data in Table 2 show clearly
that the kinetic mechanism of MM2281 is different
from the Ping Pong system of bacterial PS. The latter
consists of two half-reactions, which proceed via an
enzyme-bound pantoyl adenylate intermediate [2–5].
As a result, the pantothenate–b-alanine exchange reac-
tion of bacterial PS is independent of pantoate and
has an absolute requirement for only AMP. By com-
parison, the cosubstrate dependence of the MM2281-
catalyzed exchange reaction differs on several counts
(see above) and is inconsistent with the Ping Pong
system of bacterial PS. This shows that archaeal

Also, they are absent from the Bacteria and
Eukaryota. All of the Archaea that have archaeal-type
PS and PANK universally lack homologs to bacterial
or eukaryotic PS and PANK isoforms. In fact, bacte-
rial PS is entirely absent from the archaeal domain,
and archaeal homologs to bacterial PANK are limited
to the Thermoplasmata class.
A different situation is encountered for the first two
CoA biosynthetic steps. In the Bacteria, these steps
are catalyzed by KPHMT and KPR, which convert
a-ketoisovalerate into pantoate. A subset of the non-
methanogenic Archaea acquired these enzymes, pre-
sumably by horizontal gene transfer, from
thermophilic bacteria [10]. Individual archaeal
genomes encode either both bacterial-type KPHMT
and bacterial-type KPR or either one or none of them.
This pattern suggests that some archaeal species
produce pantoate by combining an archaeal KPHMT
isoform with bacterial-type KPR or by combining an
archaeal KPR isoform with bacterial-type KPHMT.
In other words, the distribution of bacterial-type
KPHMT and KPR genes supports the view that the
majority of the Archaea contain so far unidentified
genes that encode unrelated isoforms of KPHMT and
KPR. Moreover, the observed distribution may well
be the result of nonorthologous gene displacement
[23], where the archaeal isoforms of KPHMT and
KPR were individually replaced by their bacterial
counterparts in certain archaeal species. Verification of
this hypothesis awaits, of course, identification of the

Radiolabeled Chemicals ⁄ Biotrend Chemikalien (Cologne,
Germany). Rabbit pyruvate kinase and all other reagents
were from Sigma-Aldrich (Munich, Germany) unless indi-
cated otherwise. d-Pantoate was prepared from d-pantoyl
lactone as described elsewhere [27]. Genomic DNA from
the Me. mazei strain Goe1 (DSM 3647) was a gift from
K. Pflu
¨
ger, Universita
¨
tMu
¨
nchen.
Cloning of the Me. mazei and E. coli genes for PS
The Me. mazei ORF MM2281 (GenBank accession number
AE008384) was PCR-amplified from genomic DNA by
using Pfu polymerase (Stratagene, Amsterdam, the Nether-
lands) and the primers dGCGCGCATATGACcGATATtC
CGCACGAtCACCCGcGcTACGAATCC and dGCGCGC
TCGAGTtAGTAgCCgGTTTCCGCGGCCATGGT. The
start and stop codons are in bold. Lower-case letters desig-
nate silent nucleotide changes that were introduced to
reduce the number of rare codons for expression in E. coli.
The amplified ORF was subcloned via NdeI and XhoI
restriction sites in the primers into the pET28-a vector
(Novagen ⁄ Merck Chemicals, Darmstadt, Germany). The
resulting plasmid, pET–MM2281, contains the MM2281
ORF in translational fusion with the vector-encoded
N-terminal His-tag, leading to the expression of NH
2

–
)
The plasmids pGEM–MM2281 and pBKS–panC (positive
control) and the empty pBluescript KS– vector (negative
control) were introduced into the pantothenate-auxotrophic
E. coli strain AT1371. Single colonies of the transformants
were grown overnight at 37 °C in 5 mL of liquid dYT
medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl)
containing 100 lgÆlL
)1
ampicillin. The E. coli cells were
pelleted and washed twice in 5 mL of GB1 buffer [100 mm
potassium phosphate, pH 7.0, 2 gÆL
)1
(NH
4
)
2
SO
4
]. The
pelleted cells were resuspended in GB1 buffer, adjusted to
an D
600 nm
of 0.3, and incubated at 25 °C for 1 h. The
starved cells were then used to inoculate [0.5% (v ⁄ v)] the
experimental cultures (4 gÆL
)1
glucose, 0.25 gÆL
)1

Overexpression and purification of MM2281 and
helper enzymes
The MM2281 protein was expressed in E. coli BL21(DE3)
carrying the pET–MM2281 plasmid described above and
purified on Ni–nitrilotriacetic acid agarose (Qiagen, Hilden,
Pantothenate synthetase from Methanosarcina mazei S. Ronconi et al.
2762 FEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBS
Germany) following the manufacturer’s standard protocol.
After affinity chromatography, MM2281 was loaded onto a
MonoQ anion exchange column equilibrated in 50 mm
Tris ⁄ HCl (pH 8.8). In a linear 0–1 m KCl gradient,
MM2281 eluted at approximately 350 mm KCl. The
enzyme preparation was then dialyzed exhaustively against
50 mm Tris ⁄ SO
4
(pH 8.0) and 5 mm dithiothreitol, frozen
in liquid N
2
, and stored in aliquots at )70 °C. The native
molecular mass of MM2281 was estimated by gel filtration
chromatography as previously described [6]. E. coli PS [6]
and E. coli PANK [28] were overexpressed and purified as
previously described. Protein concentrations were deter-
mined using the Bradford protein assay kit (Bio-Rad,
Munich, Germany) with BSA as standard.
Enzyme assays
The standard assay for PS activity contained 20 mm potas-
sium d-pantoate, 1 mm b-alanine, 0.08 mm [3-
14
C]b-alanine

(1 lg) and E. coli PANK (2.5 lg) or no enzymes.
In order to detect possible adenosine nucleotide products
of MM2281, standard assays containing MM2281 alone or
together with E. coli PANK were carried out as described
above, except that [3-
14
C]b-alanine was omitted. The reac-
tion was quenched after 180 min, and the products were
cochromatographed with authentic ATP, ADP and AMP
standards (Sigma). Adenosine nucleotides were separated
on silica plates using dioxane ⁄ NH
3
(25%) ⁄ H
2
O(6:1:4)
as a mobile phase and detected under UV light (254 nm).
Isotope exchange assay
The pantothenate–b-alanine isotope exchange was assayed
at 25 °C, and the standard reaction contained 1 mm b-ala-
nine, 0.07 mm [3-
14
C] b-alanine (55 mCiÆmmol
)1
), 5 mm
pantothenate, 5 mm ADP, 5 mm sodium phosphate, 5 mm
ATP, 20 mm potassium d-pantoate, 10 mm MgSO
4
,
7.5 mm K
2

confidence in STRING) or weak links (low or medium con-
fidence in STRING).
Acknowledgements
We would like to thank Katharina Pflu
¨
ger, Universita
¨
t
Mu
¨
nchen, for genomic DNA from Me. mazei, and
Ishac Nazi, McMaster University, for the E. coli
PANK expression plasmid pPANK. We also thank
Erich Glawischnig for critically reading this manu-
script. S. Ronconi was funded by graduate scholar-
ships from the German Academic Exchange Service
(DAAD) and Technische Universita
¨
tMu
¨
nchen
(Frauenbu
¨
ro). Work on pantothenate and CoA
biosynthesis in this laboratory was funded by the
Deutsche Forschungsgemeinschaft.
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