However, there are no archaeal homologs to known isoforms of pantothenate synthetase PS or pantothenate kinase.. MM2281 also transferred the14C-label from [14C]b-alanine to pantothenate
Trang 1the Archaea
Silvia Ronconi, Rafal Jonczyk and Ulrich Genschel
Lehrstuhl fu¨r Genetik, Technische Universita¨t Mu¨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 pantopanto-thenate
in their diet
Pantothenate synthetase (PS) catalyzes the last step in the biosynthesis of pantothenic acid, also known as vita-min B5 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
to the HIGH superfamily of nucleotidyltransferases [3]
Keywords
archaeal metabolism; CoA biosynthesis;
evolution of metabolism;
Methanosarcina mazei; pantothenate
synthetase
Correspondence
U Genschel, Lehrstuhl fu¨r Genetik,
Technische Universita¨t Mu¨nchen, Am
Hochanger 8, 85350 Freising, Germany
Fax: +49 8161 715636
Tel: +49 8161 715644
E-mail: genschel@wzw.tum.de
(Received 7 February 2008, revised 17
March 2008, accepted 19 March 2008)
doi:10.1111/j.1742-4658.2008.06416.x
The linear biosynthetic pathway leading from a-ketoisovalerate to panto-thenate (vitamin B5) and on to CoA comprises eight steps in the Bacteria and Eukaryota Genes for up to six steps of this pathway can be identified
by sequence homology in individual archaeal genomes However, there are
no archaeal homologs to known isoforms of pantothenate synthetase (PS)
or pantothenate kinase Using comparative genomics, we previously identi-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 [14C]4¢-phosphopantothenate from [14C]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 the14C-label from [14C]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.
Trang 2D-pantoateþ b-alanineþATP! D-pantothenateþAMPþPPi
ð1Þ CoA biosynthesis is best understood in the Bacteria,
where eight steps lead from a-ketoisovalerate to CoA
(Fig 1) [7,8] The eukaryotic CoA pathway has been
studied to various degrees in fungi, plants, and animals
[9], and consists of both highly conserved bacterial-type
enzymes and divergent isoforms Much less is known
about CoA biosynthesis in the Archaea We previously used comparative genomics to reconstruct the universal CoA biosynthetic pathway in the Bacteria, Eukaryota, and Archaea [10] Archaeal genes for the ultimate four steps can be identified by homology in all archaeal genomes, and experimental confirmation of this assign-ment is available for three of these steps [11,12] In addition, bacterial-type genes for the first two steps are obvious in a number of the nonmethanogenic Archaea However, homologs to bacterial PS or any of the three established isoforms of pantothenate kinase (PANK) [13] are generally missing from archaeal genomes We approached this problem by using a nonhomology search strategy based on conserved chromosomal prox-imity, a method that exploits the tendency of function-ally related genes to cluster along the chromosome [14] Using the archaeal CoA biosynthetic genes with homol-ogy to bacterial or eukaryotic genes on the CoA path-way as a starting point, this identified the clusters of orthologous groups (COG)1701 and COG1829 protein families as the best candidates for the PS and PANK 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
Archaea The linear pathway leading from a-ketoisovalerate to CoA
comprises eight steps in the Bacteria and Eukaryota It proceeds
via pantoate, pantothenate (vitamin B 5 ), and
4¢-phosphopantothe-nate The remaining intermediates, as well as the branch for
pro-duction of b-alanine, are left out for clarity For six of these steps,
homologs can be established in the Archaea, and the corresponding
COG families are shown in color to indicate the average level of
sequence identity to the respective E coli or human CoA
biosyn-thetic enzymes [10] Nonhomologous functional links to the
archa-eal homologs were obtained from the STRING database [16], as
described in Experimental procedures Taken together, the links to
COG1701 and COG1829 clearly support these protein families as
the best candidates for the missing steps in archaeal CoA
biosyn-thesis The functional assignments for COG1701 (archaeal PS) and
COG1829 (archaeal PANK) are explained in the main text PPCS,
phosphopantothenoylcysteine synthetase; PPCDC,
phospho-pantothenoylcysteine decarboxylase; PPAT, phosphopantetheine
adenylyltransferase; DPCK, dephospho-Co A kinase;
P-pantothe-nate, 4¢-phosphopantothenate.
Trang 3Consistent with the assumption that both protein
families represent archaeal isoforms of CoA
biosyn-thetic enzymes, their members are found in nearly all
archaeal genomes, but not outside the archaeal
domain Furthermore, COG1701 and COG1829 share
a strictly conserved phylogenetic profile and frequently
occur in tandem in potential operons (e.g in Me
maz-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
in cultures supplemented with pantothenate (not shown) The cultures generally showed long lag peri-ods, during which the cells recovered from the starving procedure in pantothenate-free medium (see Experi-mental procedures) In the absence of supplements, AT1371 cells harboring MM2281 showed a shorter lag period and faster growth than the negative control, but did not grow as well as the positive control (Fig 3A) The same pattern was observed in minimal medium containing 1 mm pantoate, a substrate of PS, except that the growth of cells containing MM2281 or the negative control was stimulated (Fig 3B) In our hands, the E coli panC mutant carrying empty vector (negative control) showed minor growth in minimal
Fig 2 Potential operon for CoA biosynthesis in Me mazei The
predicted genes for PS and PANK, as well as the dfp gene
encod-ing the bifunctional enzyme PPCS ⁄ PPCDC (MM2281 through
MM2283), occur in a cluster, which corresponds to the steps
lead-ing from pantoate to 4¢-phosphopantetheine PPCS,
phospho-pantothenoylcysteine synthetase; PPCDC, phosphopantothenoyl
cysteine decarboxylase.
Fig 3 Functional complementation of an E coli pantothenate auxotrophic mutant The pantothenate-requiring E coli mutant car-rying the Me mazei MM2281 gene (d), the E coli panC gene (h)
or empty vector (s) was grown in liquid culture in the absence of pantothenate, as described in Experimental procedures The mini-mal medium contained no supplements (A) or an additional 1 m M
pantoate (B) All transformants grew equally well when the medium was supplemented with 1 m M pantothenate (not shown).
Trang 4medium 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 [14C]b-alanine converted into
[14C]pantothenate was below the lower limit of
detec-tion (1% conversion) This means that PS activity of
MM2281 was absent or below 0.6 nmolÆmin)1Æmg)1 in
our assay
In an attempt to confirm pantothenate as a product
of MM2281, we coupled the reaction with excess
E coli PANK, which converts pantothenate to
4¢-phosphopantothenate MM2281, individual helper
enzymes or combinations of these were assayed for
their ability to convert [14C]b-alanine into [14
C]panto-thenate or [14C]4¢-phosphopantothenate under
stan-dard conditions The 14C-labeled reaction products
were separated by TLC, revealed by phosphoimaging
(Fig 4), and quantified to calculate specific PS
activi-ties (Table 1) Whereas MM2281 alone had no
signifi-cant pantothenate-synthesizing activity in this assay, it
was obvious that E coli PS efficiently converted
[14C]b-alanine into [14C]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
[14C]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.
Table 1 Formation of pantothenate or 4¢-phosphopantothenate through MM2281 and helper enzymes MM2281 and helper enzymes were tested individually or in combinations for their ability
to form [14C]pantothenate or [14C]4¢-phosphopantothenate from [ 14 C]b-alanine, pantoate and ATP under the conditions of the stan-dard assay (Fig 4) ND, not detectable; PyrK, rabbit pyruvate kinase.
Enzyme(s)
Specific pantothenate synthetase activity (nmolÆmin)1Æmg)1) [14C]
Pantothenate
[14C]
4¢-Phosphopantothenate
E coli PS + E coli PANK ND > 900 a
MM2281 + E coli PANK ND 94 ± 11b
MM2281 + PyrK +
E coli PANK
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.
Trang 5thenate 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)1Æmg)1 with respect to
MM2281, indicating that the E coli PANK-mediated
removal of pantothenate accelerated the synthesis of
pantothenate through MM2281 at least 100-fold
Principally, the above behavior can be explained by
assuming either that MM2281 is potently inhibited by
pantothenate or that equilibrium is reached after only
a small fraction of substrates has reacted In both
cases, the reaction is expected to accelerate when
pan-tothenate is removed, because this should abolish
inhi-bition or displace the equilibrium It should be
mentioned that active removal of pantothenate has no
significant effect on the reaction rate of bacterial PS,
essentially excluding the possibility that the MM2281
preparation was contaminated with bacterial PS This
is because pantothenate was shown to be a very weak
product inhibitor of My tuberculosis PS [4] and the
equilibrium of the reaction catalyzed by bacterial PS
lies far on the product side The latter statement can
be derived by considering the equilibrium constant of
the reaction catalyzed by bacterial PS (Eqn 1) The
equilibrium constant for Eqn (1) has not been
deter-mined experimentally, but can be deduced from the
equilibrium constants for the hydrolysis of
pantothe-nate into pantoate and b-alanine (K¢ = 42 at pH 8.1
and 25C [20]) and the phosphorolysis of ATP into
AMP and PPi (K¢ = 3 · 109 at pH 8 and 25C [21])
Combining the above constants gives the overall
equi-librium constant for Eqn (1) at approximately pH 8
and 25C (K¢Eqn (1)= 7.2· 107) The large value
means that the reaction in Eqn (1) will go to
comple-tion under physiological condicomple-tions, including the
enzyme assay used in this study (pH 8.0, 37C)
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-son, stoichiometric coupling of E coli PANK, which produces ADP, with a bacterial, AMP-forming PS would lead to the accumulation of equimolar amounts
of ADP and AMP The observations that synthesis of phosphopantothenate through MM2281 and E coli PANK was accelerated by removing ADP and not accompanied by production of AMP gave rise to the hypothesis that MM2281 is an ADP-forming synthe-tase according to Eqn (2):
D-pantoateþ b-alanineþATP! D-pantothenateþADPþPi
ð2Þ The equilibrium constant for Eqn (2) can be calcu-lated in the same way as that for Eqn (1) (see above) Using the equilibrium constant for phosphorolysis of ATP into ADP and Pi (K¢ = 1.6 · 107 at pH 8 and
25C [21]), the overall equilibrium constant for Eqn (2) at approximately pH 8 and 25C becomes
Fig 5 Detection of adenosine nucleotides produced by MM2281 (Me mazei PS) and E coli PANK Standard assays containing MM2281 alone (lane 4) or together with E coli PANK (lane 5) were carried out as described in Experimental procedures Reaction mix-tures were separated by TLC, and nucleotides were visualized under
UV light Authentic standards of ATP, ADP and AMP were cochro-matographed on the same plate (lanes 1–3) EcPANK, E coli PANK.
Trang 6K¢Eqn (2)= 3.9· 105 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 novosynthesis 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-ucts (complete system) or, if the enzyme catalyzes a
partial reaction, in the presence of a subset of
reac-tants Each cosubstrate may either be indispensable for
the exchange reaction to occur or merely affect the
exchange rate [22]
The MM2281-catalyzed incorporation of 14C-label from [14C]b-alanine into pantothenate was investigated
in the presence of full sets or subsets of the reactants
in Eqn (1) or Eqn (2), respectively (Table 2, Experi-ment I) In the presence of the full set of reactants (complete system), the assay based on Eqn (2) revealed
a five-fold higher rate than that based on Eqn (1) Removing pantoate reduced the incorporation of
14C-label into pantothenate to negligible levels in both the Eqns (1,2) systems In contrast, removing ATP abolished the accumulation of [14C]pantothenate only
in the Eqn (1) system, whereas the Eqn (2) system retained approximately 50% exchange activity This means that the pantothenate–b-alanine exchange in the Eqn (2) system has no absolute requirement for ATP, and this result was confirmed by a second set of iso-tope exchange assays (Table 2, Experiment II) When inorganic phosphate (Pi) 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 Pi are dispensable However, when both ATP and ADP were removed, the resulting exchange activity was negligible In summary, MM2281 catalyzed significant transfer of 14C-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
MM2281 alone showed no detectable net synthesis
of pantothenate in the forward assay (see above; Fig 4), and the forward rate would be expected to be even lower in the presence of products We assume, therefore, that the transfer of14C-label from b-alanine
to pantothenate was due to isotope exchange Maximal exchange activity occurred in the complete system of Eqn (2), pointing to Eqn (2) as the basic reaction for MM2281 Moreover, our data suggest that MM2281
is able to catalyze an ADP-dependent, but not an AMP-dependent, pantothenate–b-alanine exchange
Table 2 MM2281-catalyzed isotope exchange between [ 14
C]b-ala-nine and pantothenate MM2281 was assayed for its ability to
transfer 14 C-label from [ 14 C]b-alanine to pantothenate in the
pres-ence or abspres-ence of cosubstrates The cosubstrates in the complete
system are ATP, AMP, PP i , and pantoate (Eqn 1), or ATP, ADP, P i ,
and pantoate (Eqn 2) Different preparations of MM2281 were used
in two independent experiments (Experiments I and II) Missing
val-ues indicate that the cosubstrate combination indicated was not
tested ND, not detectable.
Reactants
Initial exchange ratea(%) Experiment I Experiment II Eqn (2)
Minus ATP, minus pantoate 3
Eqn (1)
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)3Æmin)1 in Experiments I and II,
respectively.
Trang 7reaction in the presence of pantoate This is difficult to
reconcile with Eqn (1), and further supports Eqn (2) as
the overall reaction catalyzed by MM2281 The
resid-ual exchange activity in the complete system of
Eqn (1) may be due to contamination of the
commer-cial ATP preparation with ADP
We also considered the theoretical possibility that
MM2281 is a hydrolase, which facilitates the
equilib-rium between pantoate, b-alanine and pantothenate
according to Eqn (3):
D-pantoateþ b-alanine$ D-pantothenate ð3Þ
Although there is no simple argument to rule out
Eqn (3) as the basic reaction of MM2281, explaining
the overall behavior of MM2281 in this way is very
difficult and also generates a conflict with the reported
value for K¢Eqn (3) Most importantly, Eqn (3) implies
that ATP and ADP are not substrates of MM2281
The observed effects of ADP and ATP on the isotope
exchange rate can then be explained by assuming that
ADP and ATP effectively promote a switch from
inac-tive to acinac-tive enzyme However, this is at odds with
the observation that enzymatic removal of ADP
accel-erated MM2281 in the forward direction (see above)
Second, considering the equilibrium constant of
Eqn (3) (K¢Eqn (3)= 1⁄ 42 at pH 8.1 and 25 C [21]),
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 14C-label associated with
panto-thenate at equilibrium would be 35% However, we
observed that [14C]pantothenate accumulated up to
60% of the total 14C-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
conversion of pantoate into 4¢-phosphopantothenate
The protein families COG1701 and COG1829 were
then identified as the best candidates for the missing
steps by comparative analysis of the 16 completely
sequenced archaeal genomes available at the time [10]
The STRING database, which currently integrates 26
archaeal genomes, revealed additional functional asso-ciations that support a role for COG1701 and COG1829 in archaeal CoA biosynthesis (Fig 1) On the basis of distant homology relationships, we tenta-tively assigned the PS and PANK functions to the COG1701 and COG1829 protein families, respectively Three lines of experimental evidence support the computational prediction of archaeal PS First, the cloned COG1701 member from Me mazei (MM2281) partially complemented the auxotrophic phenotype of
an E coli mutant lacking PS activity (Fig 3) Second, the recombinant proteins MM2281 and E coli PANK together facilitated the synthesis of 4¢-phosphopantoth-enate from pantoate, b-alanine, and ATP (Fig 4) Arguably, the enzyme preparations were not contami-nated with bacterial PS, allowing the conclusion that pantothenate synthesis in the coupled assays was due
to MM2281 Third, MM2281 catalyzed the transfer of
14C-label from b-alanine to pantothenate, presumably
by isotope exchange, in a cosubstrate-dependent man-ner (Table 2) Given that function is typically con-served within orthologous groups [15], demonstration
of PS activity for one member of the group provides 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
Trang 8exchange data can readily be accounted for by Eqn (2)
but not by Eqn (1)
The highest PS activity of MM2281 observed in this
study was 140 nmolÆmin)1Æmg)1, which is equivalent to
a turnover of 0.07 s)1 This is significantly below
typi-cal values for kcat, which range from about 0.8 s)1 to
2· 105s)1 [22] More specifically, the MM2281
activ-ity reported here was more than 15-fold lower than
that of E coli PS under optimal conditions (calculated
from data in [2]) and nearly 50-fold lower than the kcat
reported for My tuberculosis PS [4] Given the
com-paratively low activity of MM2281, it is possible that
the enzyme requires an activator or cofactor that was
absent from the standard assay One attractive
candi-date for this role is the predicted PANK in Me mazei
(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
Pi 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
PS evolved a distinct mechanism to synthesize
pantothenate
Evolution of phosphopantothenate biosynthesis
We hypothesize that the entire upstream portion of
CoA biosynthesis, leading from common precursors to
phosphopantothenate, evolved independently in the
Bacteria and Archaea This view was initially based on
the finding that many archaeal genomes contain no
homologs to any of the corresponding bacterial enzymes and on the prediction of distinct archaeal forms of PS and PANK [10] The experimental evidence in this study provides strong support for the predicted identity of archaeal PS (COG1701) This, in turn, also supports the prediction of archaeal PANK (COG1829), because there are strong nonhomologous links between the two families (Fig 1)
The linear pathway from a-ketoisovalerate to phos-phopantothenate comprises four steps in the Bacteria (Fig 1) So far, unrelated archaeal genes on this path-way have been computationally predicted for the third and fourth steps (i.e PS and PANK), but not for the first and second steps [i.e ketopantoate hydroxymeth-yltransferase (KPHMT) and ketopantoate reductase (KPR)] Interestingly, the phyletic distribution of PS genes indicates that they were not subject to horizontal transfer, in either direction, between the archaeal and bacterial domains Archaeal PS and PANK isoforms show strict co-occurrence and are present in most of the Archaea, except in the Thermoplasmata class of the Euryarchaeota and in Nanoarchaeum equitans 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 archaeal genes for pantoate synthesis
Trang 9Given that horizontal gene transfer occurred
exten-sively between the thermophilic Bacteria and Archaea
[24,25], the question arises of why KPHMT and KPR
genes were transferred, whereas PS genes were not
The archaeal PS (MM2281) characterized in this study
produces pantothenate from the same precursors as
bacterial PS but has clearly distinct kinetic properties
The most striking difference is the apparent inhibition
of MM2281 by pantothenate, raising the possibility
that this step has a role in regulating archaeal CoA
biosynthesis In contrast, the most important control
point in bacterial CoA biosynthesis is PANK [9], and
no regulatory function is known for PS It is attractive
to speculate, therefore, that horizontal gene transfer
of PS, and possibly PANK, was suppressed by
incom-patible regulatory properties
Experimental procedures
Materials
tsx-29, glnV44(AS), galK2(Oc), LAM-, Rac-0, hisG4(Oc),
rfbD1, xylA5, mtl-1, argE3(Oc), thi-1] [26] was obtained
from the E coli Genetic Stock Center, Yale University
[3-14C]b-alanine (55 mCiÆmmol)1) was from American
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¨t Mu¨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
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 NH2
-MGSSHHHHHHSSGLVPRGSH-MM2281
For functional complementation of the E coli panC
mutant (AT1371), the MM2281 ORF was reamplified from
pET-MM2281 using the primers dGCGCGAGAAGGAG ATATACCATGACCGATATTCCGCACGATCACCCGC
GC and dGCGCGCTCGAGTTAGTAGCCGGTTTCCG CGGCCATGGT The ribosome-binding site in the for-ward primer is underlined, and the start and stop codons are in bold The PCR product was inserted into the pGEM-T vector (Promega, Mannheim, Germany), and a clone carrying the MM2281 ORF in the correct orientation for expression under the lac promoter was selected and named pGEM–MM2281 The E coli panC ORF was amplified from genomic DNA of E coli strain XL1Blue
dCGCGCCTCGAGGAGGAGTCACGTTATGTTAATTA TCGAAACC and dGCGCGTCTAGATTACGCCAGCTC GACCATTTT The PCR product was inserted into pBlue-script KS (Stratagene) via the restriction sites XhoI and XbaI The resulting plasmid (pBKS–panC) harbors the panCgene under the control of the lac promoter and served
as a positive control in the functional complementation experiment Automated DNA sequencing of the inserts
in pET–MM2281, pGEM–MM2281 and pBSK–panC con-firmed the desired sequences
Functional complementation of E coli AT1371 (panC–)
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
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 (NH4)2SO4] The pelleted cells were resuspended in GB1 buffer, adjusted to
an D600 nm of 0.3, and incubated at 25C 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 MgSO4.10H2O, 0.25 mgÆL)1 FeSO4.7H2O, 5 mgÆL)1 thia-mine, 68 mgÆL)1adenine, 127 mgÆL)1l-arginine, 16 mgÆL)1
l-histidine, 230 mgÆL)1 l-proline, and 100 mgÆL)1
shaking For each transformant, three cultures were started that contained an additional 1 mm pantothenate, 1 mm pantoate, or no further supplements, respectively D600 nm was determined over an incubation time of 24 h
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,
Trang 10Germany) 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⁄ SO4 (pH 8.0) and 5 mm dithiothreitol, frozen
in liquid N2, 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 [3-14C]b-alanine
K2SO4, 5 mm dithiothreitol, 50 mm Tris⁄ SO4(pH 8.0) and
2.5 lg of MM2281 in a final volume of 25 lL The reaction
was initiated by the addition of substrates, and incubated
at 37C, and 5 lL aliquots were removed at 30, 90 and
180 min time points Separation of reaction products
(10 nCi aliquots) by TLC, quantitation of14C-label above
nonenzymatic activity and estimation of initial rates was as
previously described [6] The detection of14C-label was
lin-ear between 0.1 and 20 nCi, covering a range of 1–100% of
the amount analyzed per time point The assay was carried
out in the absence or presence of E coli PANK (2.5 lg) or
pyruvate kinase from rabbit (2 units) Phosphoenolpyruvate
(2 mm) was included in the assay when pyruvate kinase was
present Control reactions in the absence of MM2281
con-tained either or both of the helper enzymes E coli PS
(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 [3-14C]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⁄ NH3 (25%)⁄ H2O (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 25C, and the standard reaction contained 1 mm
b-ala-nine, 0.07 mm [3-14C] b-alanine (55 mCiÆmmol)1), 5 mm
pantothenate, 5 mm ADP, 5 mm sodium phosphate, 5 mm
7.5 mm K2SO4, 5 mm dithiothreitol, 50 mm Tris⁄ SO4
(pH 8.0), and 1.1 lgÆlL)1 MM2281 Individual reactions
contained all of the above components [Eqn (2), complete system] or lacked one or more of the reactants ATP, ADP, pantoate, or sodium phosphate A second set of exchange reactions was carried out with AMP and sodium pyrophos-phate replacing ADP and sodium phospyrophos-phate in the above scheme [Eqn (1), complete system] Aliquots were removed from the reactions at 7, 24 and 48 h after initiation by the addition of MM2281 Quantitation of 14C-label associated with pantothenate and b-alanine and estimation of initial exchange velocities was as previously described [6]
Nonhomologous functional links
In order to verify the prediction of COG1701 and COG1829 as the missing steps in archaeal CoA biosynthesis [10], the STRING database [16] was searched for nonho-mologous functional links using the criteria ‘Neighborhood’ (conserved chromosomal proximity) and ‘Co-occurrence’ (conserved phylogenetic profile) To this end, the archaeal members of the protein families COG0413, COG1893, COG0452, COG1019, and COG0237, which are implied in archaeal CoA biosynthesis through homology (Fig 1), were used as protein queries Functional links to COG1701 or COG1829 were classified as strong links (high or highest 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¨t Mu¨nchen (Frauenbu¨ro) Work on pantothenate and CoA biosynthesis in this laboratory was funded by the Deutsche Forschungsgemeinschaft
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