BACKGROUND
our laboratory runs a structural genomics project  targeting fungal protein-coding genes in search of new anti-fungal targets.
the main goal of this project is to quantitatively express the selected saccharomyces cerevisiae and candida albicans genes products to characterize them functionally and structurally as well as explore their potential as new drug targets.
the genes selected in candida albicans are of two types: 1) they have orthologous genes in saccharomyces cerevisiae that are essential or 2) they are conserved in pathogenic fungal genomes and can be absent from the non-pathogenic yeast saccharomyces cerevisiae.
candida albicans is a diploid organism with eight sets of homologous chromosomes and a genome size of about  <dig> mb.
this pathogen is part of the normal microflora of the human gastrointestinal tract or oropharynx and is responsible for various nosocomial infections, potentially lethal in immunocompromised patients or patients in the intensive care unit.
the ca <dig> protein is encoded by a gene conserved across all known fungal genomes.
it shares 36% sequence identity with saccharomyces cerevisiae yor143c  <cit> , a thiamine pyrophosphokinase  essential for fungus growth and survival  <cit> .
this enzyme is also essential in s. pombe  <cit> .
the production of thiamine pyrophosphate , catalyzed by tpk, is critical for both catabolic and anabolic cellular processes.
in vertebrates, a deficiency in thiamine synthesis is linked to neurological diseases such as beriberi and wernicke encephalopathy  <cit> .
in all synthesizing organisms, thiamine  is produced from phosphorylated het thiazole) and hmp  that are condensed into thiamine phosphate.
in yeast, this thiamine phosphate is then hydrolyzed into thiamine before pyrophosphorylation by the tpk that catalyzes the transfer of a pyrophosphate  from atp to thiamine, resulting in amp and tpp  <cit> .
this article reports the enzymatic characterization as well as the structure determination of the candida albicans ca <dig> thiamine pyrophosphokinase in complex with both thiamine and thiamine-pnp .
we also present a comparison of ca <dig> with the structure of the mouse tpk ternary complex with pyrithiamine pyrophosphate and amp   <cit> , with the tpk s. cerevisiae structure   <cit>  and with sequences from other species.
methods
general cloning strategy
the cdnas corresponding to the tpk genes were amplified by pcr from their respective genomic dnas .
gene cloning was performed using the ligation-independent cloning  method based on ligation of sticky ends generated by t <dig> dna polymerase  <cit> .
for the purpose of the profun project, we constructed a specific expression vector, psf- <dig>  patent  <cit> , compatible with lic cloning using the following procedure.
the ecori+bamhi region of pqe <dig> was inserted in pqe80l vector where xhoi, ncoi and mfei sites have been mutated.
then the lacz encoding gene was pcr amplified using primers containing flanking sequences  used for lic cloning:
5'-ccatggctcatcaccatcaccatcacgggcatcaccatcaattg, forward primer containing the coding sequence  for a poly-histidine tag,
5'-ggatccctcgagttagtcaccatccaattg, reverse primer.
in the absence of an insert, the psf- <dig> expression vector expresses the lacz gene, while the in frame insertion of the resulting pcr products into ncoi+bamhi sites deactivates the gene thus allowing the easy detection of the parental vector  <cit> .
in addition; the gfp encoding gene was pcr-amplified using primers containing flanking sequences 5'-ctcgaggcggccg and 5'-ggatccattatgcggccgc.
this pcr product was inserted at the 3'end of the lacz encoding gene into xhoi+bamhi sites yielding the psf- <dig> expression vector that expresses the gfp in c-terminal fusion with the targeted gene.
not <dig> restriction sites  flanking the gfp encoding gene allows its removal  <cit> .
cloning and expression screening of ca <dig> and yor143c tpk genes
pcr amplifications were performed using primers specific for ca <dig> and yor143c tpk genes preceded by 5'-catcaccatcaattg  and 5'-tcaccatccaattg  together with purified candida albicans and saccharomyces cerevisiae genomic dna as template.
the pcr products were directly purified using the nucleospin extract kit .
then  <dig>  pmol of the purified pcr product was treated with t <dig> dna polymerase in the presence of  <dig>  mm of dctp for  <dig> min.
at 22°c before inactivating the enzyme by heating  <dig> min at 75°c.
in a parallel procedure, the expression vector, psf- <dig>  was digested with the mfel restriction enzyme to excise the insert bearing the lacz encoding sequence.
psf- <dig> was then purified on agarose gel using the nucleospin extract kit  and treated with t <dig> dna polymerase in the presence of  <dig>  mm of dgtp for  <dig> min at 22°c before inactivating the enzyme by heating  <dig> min.
at 75°c.
the cloning step of the two fungi tpk genes consisted in a hybridization reaction performed by mixing  <dig>  pmol of psf- <dig> and  <dig>  pmol of the insert in a reaction volume of  <dig> μl and an incubation time of  <dig> min.
at 22°c,  <dig> μl of  <dig> mm edta was then added to the reaction mixture.
after a second incubation period of  <dig> min.
at 22°c, the entire hybridization reaction was used to transform e. coli dh5α.
selection was performed on lb plates containing  <dig> μg/ml ampicillin, positive plasmids were then isolated.
this cloning procedure allowed insertion of the gene of interest in frame with a sequence encoding the n-terminal  <dig> tag and a ghhhql sequence corresponding to the translation of the forward primer sequence.
an additional c-terminal fragment qldgdleaa corresponds to the translation of a linker between the gene of interest and the sequence encoding the gfp.
an expression screening was performed using our standard procedure  <cit>  and the gfp gene reporter was used to quantify the soluble expression through fluorescence measurements  <cit>  in order to identify the best condition for the fungi tpk proteins soluble expression.
subsequent removal of the gfp encoding gene was achieved by digesting the plasmid by noti followed by intra ligation.
as a result, the plasmids carrying the ca <dig> gene were over-expressed in e. coli origami in 1l flasks containing sb medium cultured overnight at 17°c.
induction was performed using iptg  when the culture reached an od <dig> nm of  <dig> .
the plasmids carrying the yor143c gene were expressed in e. coli origami in 1l flasks containing 2ytg medium at 25°c after induction with iptg  when the culture reached an od <dig> nm of  <dig> .
after centrifugation, the pellets were resuspended in buffer a  with  <dig> % glycerol and  <dig>  % triton x- <dig> then sonicated and centrifuged again.
purification
the cleared lysate was applied to a  <dig> ml hitrap chelating column  loaded with ni2+ and equilibrated with buffer a. the column was washed with  <dig> column volumes of buffer a,  <dig> column volumes of buffer a containing  <dig> mm imidazole and  <dig> column volumes of buffer a containing  <dig> mm imidazole at a flow rate of  <dig> ml.min- <dig>  elution was performed with a linear gradient over  <dig> column volumes from  <dig> mm to  <dig> mm imidazole.
the fractions corresponding to the elution of ca <dig> and yor <dig> tpk proteins with 150– <dig> mm imidazole were run on a desalting column  and analyzed by mass spectroscopy and n-terminal edman sequencing.
after purification, the fractions contained at least 98% pure protein in tris buffer  <dig> mm ph  <dig>  and tris buffer  <dig> mm,  <dig> mm nacl ph  <dig> .
for the ca <dig> protein, the isoelectrofocalisation revealed a band around  <dig>  instead of the predicted pi of  <dig> .
gel filtration of the purified tpk proteins on a sephacryl s <dig> hr column indicated the final products were dimeric in solution.
enzymatic assays
in order to verify that our two c. albicans and s. cerevisiae enzymes were active tpk we developed a protocol involving four consecutive reactions and different enzymes.
the tpk activities were determined at room temperature using purified protein  in tris buffer  in presence of  <dig> mm thiamine and  <dig> mm atp.
the reaction cascade was monitored through the nadh decrease in absorbance at λ =  <dig> nm.
the first reaction is catalyzed by the tpk and produces a tpp and an amp.
the myokinase then uses the produced amp in the presence of atp to produce  <dig> adp molecules, each of them being used by the pyruvate kinase enzyme in presence of phosphoenol pyruvate  to produce a pyruvate and an atp molecule.
the last step is catalyzed by the lactate dehydrogenase hydrolyzing pyruvate in the presence of nadh to produce l-lactate and nad+.
crystallisation
the c. albicans tpk recombinant protein was concentrated to  <dig>  g/l in  <dig> mm tris buffer at ph  <dig>  using a centrifugal filter device .
precipitation experiments were carried out at  <dig> k using various precipitating agents  at various phs  to determine the optimal protein concentration for crystallization.
the screening for crystallization conditions was performed on  <dig> × 96-well crystallization plates  loaded by an 8-needle dispensing robot , using a  <dig> μl sitting drop per condition at  <dig> k. the tested  <dig> crystallization conditions include in-house designed  <cit>  and commercially available solution sets .
the tpk protein  was then incubated with amp-pnp  and/or thiamine  for few minutes and the best crystals of each protein/ligand complex were obtained using the hanging drop vapor diffusion method with a  <dig> ml reservoir.
crystallization droplets were made of  <dig>  μl of complex mixed with  <dig>  μl of the reservoir solution made of  <dig>  to  <dig> % peg <dig>  mgcl <dig>  <dig>  m, tris  <dig>  m,  <dig> % glycerol between ph  <dig>  and  <dig> .
crystals appeared within a few days.
data collection
crystals of the tpk protein were collected in a hampton research  <dig>  mm <dig> loop, flash frozen to  <dig> k in a cold nitrogen gas stream and subjected to x-rays.
a first data set was collected from a crystal of tpk incubated with thiamine on a marccd  camera at the european synchrotron radiation facility  on the bm30a-fip beamline at a wavelength of  <dig>  Å. a second data set corresponding to a crystal of tpk incubated both with thiamine and amp-pnp was collected at esrf on id <dig> beamline on an adsc q <dig> 2d detector at a wavelength of  <dig>  Å. the diffraction data were indexed with mosflm  <cit>  and scaled with the scala  <cit>  software from the ccp <dig> suite  <cit> .
the two crystals belong to the space group p <dig> with two molecules in the asymmetric unit.
all statistics of the processed data are summarized in table  <dig>
<dig> values in parentheses are for the highest resolution shell.
<dig> < i/σi>, is the mean signal to noise ratio, where i is the integrated intensity of a measured reflection and σ is the estimated error in the measurement.
<dig> rsym = ∑h ∑i|ih, i - ⟨ih⟩|/∑h ∑i|ih, i, where i is the integrated intensity of reflection h having i observations and ⟨ih⟩ is the mean recorded intensity of reflection h over multiple recording.
<dig> rcryst = ∑||fo|-|fc||/∑|fo|, where fo are observed and fc calculated structure factor amplitudes.
rfree is calculated from a randomly chosen  <dig> % of reflections.
structure determination
the c. albicans tpk crystal structure in complex with thiamine has been determined by molecular replacement using the caspr server  <cit> .
to generate the c. albicans tpk models, we used the available three-dimensional structures of saccharomyces cerevisiae and mus musculus thiamine pyrophosphokinase  as template and tpk related sequences from other species .
modeller produced  <dig> models which were screened for a molecular replacement solution using  <dig> to  <dig> Å data and the amore software  <cit> .
the space group was first misinterpreted as c <dig> with one monomer per asymmetric unit and cell parameters of a =  <dig>  b =  <dig>  c =  <dig> , β =  <dig> .
the structure was solved in this space group.
the best model produced a solution with one monomer and a correlation of  <dig> % and a r-factor of  <dig> % prior to cns refinement.
after one round of rigid-body refinement and minimization using the cns program  <cit>  with  <dig> to  <dig> Å data, the r-factor dropped to 42% with a 50% rfree.
rapidly, the refinement stopped converging and we suspected wrong symmetry assignation.
we thus reprocessed the tpk/thiamine/amp-pnp data in p <dig> space group and computed a self-rotation function using amore which resulted in a peak corresponding to a two-fold pseudo symmetry with a correlation coefficient of  <dig> % using data between  <dig> and  <dig> Å thus confirming the p <dig> space group.
the two monomers, related by a non crystallographic symmetry , mainly present differences in flexible loops and water molecules.
the model was manually corrected using turbo-frodo  <cit>  followed by several rounds of minimization and individual b-factors refinements using  <dig>  to  <dig>  Å data and the cns software.
<dig> water molecules were built into the model as well as a thiamine-pnp molecule, three mg2+ and one cl- ion per monomer.
some residues were still disordered in loops l <dig>  l <dig> and l <dig> and are absent from the deposited structure.
the final free and working r-values are  <dig> % and  <dig> % respectively.
the refined structure was then used against the tpk/thiamine data and refinement was performed using several manual rebuilding cycles followed by minimization and b-factor refinement using coot  <cit>  and refmac  <cit> .
<dig> water molecules were built into this model as well as a thiamine molecule and two mg2+ ions per monomer.
again, some residues were still disordered in loops l <dig>  l <dig>  l <dig> and l <dig> and are absent from the deposited structure.
the final free and working r-values are  <dig> % and  <dig> % respectively.
RESULTS
tpk activity of the c. albicans recombinant protein
the activity of the s. cerevisiae tpk protein was previously measured on the native protein extracted from yeast  <cit> .
to validate our indirect enzymatic assay, we used the s. cerevisiae enzyme as a tpk activity control.
under the conditions described in the methods section we measured specific activities of  <dig>  nm/min/mg for the s. cerevisiae enzyme  and of  <dig>  nm/min/mg for the c. albicans recombinant protein.
the ca <dig> overall structure: monomer
as expected, the overall structure of the candida albicans tpk is similar to other members of the thiamine pyrophosphokinase family.
ca <dig> is a homodimer, with each monomer constituted of two domains.
the d <dig> domain  is an αβ fold with a twisted β-sheet made of six parallel strands  surrounded by six helices .
the d <dig> domain is a β sandwich composed of two layers arranged in a jelly-roll topology .
antiparallel strands β  <dig>  β  <dig>  β  <dig>  β 13-β  <dig>  β  <dig>  and β  <dig> parallel to β <dig> constitute the first layer.
antiparallel strands β  <dig>  β  <dig>  β  <dig> and β  <dig> constitute the second one.
both domains contribute to the dimeric association with a  <dig> Å <dig> buried surface area.
we used the 3d-coffee server  <cit>  to perform a structural alignment of  <dig> selected tpk sequences of various origins using ca <dig>  saccharomyces cerevisiae   <cit>  and mus musculus   <cit>  tpk as structural templates.
despite the high variability among these tpk sequences , there is strong residue conservation around the catalytic center .
four aspartates are strictly conserved  and have already been proposed to be involved in the stabilization of the atp molecule through mg2+ contacts  <cit> .
an aromatic residue is always found at position  <dig>  and is known to participate in the thiamine stabilization  <cit> .
an asparagine residue  is strictly conserved but its function is not yet understood.
this residue is close enough to the thiamine substrate to make some contacts with its amidopyrimidine ring.
interestingly, there are three loops in the c. albicans tpk structure.
two of them, l <dig> and l <dig> , are unique to c. albicans, while the third and longest insert  is also present in the yeast homolog with which it shares less than 20% identity over  <dig> residues .
none of these fragments appear to be directly involved in the catalytic reaction or in the ligands binding .
in the structure, the l <dig> loop lies in the solvent, in an open conformation and away from the rest of the molecule .
the b-factors for this loop are also very high .
we first built an approximate loop by following the residual density present in the 2fo-fc and fo-fc maps.
we then used manual docking to verify that the length and geometry of this loop was compatible with a transient interaction with the ligands.
this loop may thus play a role during the binding of the substrates, or in the pyrophosphorylation mechanism itself.
the ca <dig> overall structure: dimer
the main differences between the two monomers related by the ncs involve flexible loops disordered in the crystal structure .
they include loops l <dig>  l <dig> and l <dig>  which are too distant to be involved in the active site formation and located too far from symmetry related molecules to possibly be directly involved in the crystal packing.
there are also water molecules which are not strictly symmetrical between the two monomers .
although the loop l <dig> is highly mobile and associated with high b-factors  it presents little differences between the two monomers.
the l8-α5-l <dig> fragment, which is involved in crystal packing through interactions between l8-α <dig> helix/turn and α3-β <dig> helix/turn in the two monomers, can be superposed with less than  <dig>  Å root mean square deviation  based on α carbon  superposition.
there are no major differences between the tpk known structures based on cα superposition using lsqman  <cit> .
rmsd are  <dig>  Å  between the c. albicans and the s. cerevisiae tpk structures, and  <dig>  Å  between the c. albicans and the mouse tpk.
the backbone trace around the thiamine binding site superimposes well except for one fragment.
timm et al.
described a rearrangement of the l <dig> loop in the mouse tpk when comparing the structures with thiamine and tpp  <cit> .
this rearrangement is not observed within the c. albicans tpk which fits the mouse tpk in complex with tpp except for the s <dig> side chain.
thiamine binding site
two binding sites can be shown in the asymmetric unit.
the strong electronic density is consistent with thiamine molecules present at the thiamine binding site .
as in the mouse structure, the thiamine moieties are located at an extremity of the groove formed by the dimer assembly.
for convenience, we will take a as the reference monomer and b as the second one  but all observations made on one of the active sites can be transposed to the second one.
the ligands are maintained by both main chains and side chains interactions.
these interactions involve the strands β <dig>  β <dig>  β <dig>  of monomer b and the loops l <dig>  l <dig> of monomer a.
as observed in other structures  there is a weak stacking between an aromatic residue  and the amidopyrimidine ring .
the cm <dig> carbon makes hydrophobic interactions with the w290b side chain, a residue conserved in the sequence of the s. cerevisiae homologue.
in the mouse sequence this residue is replaced by an aspartate and does not make any contact with the ligand.
nevertheless, a certain hydrophobic environment is maintained in the murine structure with the spatial proximity of a leucine .
the thiamine ammonium group n <dig> of the amidopyrimidine ring is engaged in a salt bridge with the q138a main-chain carbonyl oxygen .
the opposite side of this ring is stabilized by another salt bridge involving n <dig> and the oxygen from the s299b side chain .
the aspartate residue interacting with n <dig> and n <dig> described in the mouse structures  <cit>  is replaced by a tyrosine  too far away from the ring to interact with it.
n <dig> from the thiazolium ring is stabilized by electrostatic interactions between the main chain carbonyl oxygen atoms of s300b and q138a.
at least  <dig> residues involved in the thiamine binding pocket  are poorly conserved between different species but well represented in fungus  and could be the focus of specific antifungal drug design.
magnesium binding site in the tpk/thiamine complex
as observed in the mouse tpk/tpp complex, an mg2+ ion is maintained by electrostatic interactions with the side chain of the four strictly conserved aspartate residues .
the  <dig> last coordinations are made with water molecules  <dig> and  <dig> .
timm et al.
highlighted that a second mg2+ ion could be involved in the reaction process as in another pyrophosphokinase .
interestingly, the c. albicans tpk/thiamine complex reveals a second mg2+ partially stabilized by the hydroxyl group o <dig> of thiamine and a salt bridge with d <dig> and d <dig> side chains and with two main chain carboxy oxygens .
the nitrogen atom from the k <dig> side chain participates in the last coordination .
as proposed by timm et al., this lysine could be involved in the activation of the thiamine hydroxyl by the stabilization of a water molecule  <cit>  or, as in this c. albicans structure, of a magnesium ion.
thiamine-pnp binding site, implications for the atp binding site
in order to compare the c. albicans and mouse tpk binding sites, we used  <dig> atp analogues  during the crystallization process.
in the amp-cpp molecule a carbon atom replaces the oxygen atom located between the α and β phosphates in the atp molecule .
in the amp-pnp molecule, a nitrogen atom  replaces o <dig>  while we did not get crystals of the tpk/thiamine/amp-cpp complex, the tpk incubated with thiamine and amp-pnp produced usable crystals.
however, the structure revealed that instead of a tpk/thiamine/amp-pnp complex, we obtained an unexpected tpk/thiamine-pnp complex with a tpnp molecule in the enzyme binding site .
this result is consistent with the earlier suggestion that the pyrophosphate group is transferred at once from the atp molecule to the thiamine moiety.
the superimposition of the c. albicans tpk/tpnp complex onto the mouse tpk/tpp complex  highlights few differences between the two thiamine phosphate ends.
the nitrogen n <dig> in the tpnp molecule is symmetrically opposed to its equivalent oxygen o <dig> in the tpp molecule.
it is stabilized by a weak electrostatic interaction  with the s301b γ-oxygen in the c. albicans tpk/tpnp complex .
in the mouse tpk/tpp complex, this serine residue, located in the amp binding pocket, is at  <dig>  Å from the o <dig>  we propose that this serine residue could repulse the tpp in normal conditions and help to release it out of the active site.
in the mouse tpk/tpp complex, q134a interacts directly with the phosphate end of the thiamine.
the equivalent glutamine  adopts the same conformation in the c. albicans tpk/thiamine complex and an opened conformation in the c. albicans tpk/tpnp complex.
in the mouse tpk complex with amp and tpp  <cit> , the atp binding site involves residues poorly conserved across different species.
in the c. albicans tpk structure, no extra density that could correspond to a nucleotide was found even if this site is opened enough to accommodate an atp molecule.
interestingly, exactly opposed to the mouse amp binding site, , we have identified an inorganic phosphate  .
this phosphate is stabilized by a glutamine , an arginine  and a salt bridge involving a magnesium ion .
moreover, despite the lack of strong residual density, an amp molecule could reasonably be accommodated at that position with an average b-factor of  <dig> and a good occupancy .
however, the non-phosphate part of this putative amp molecule presents higher b-factors  and interacts with side chains of poorly conserved residues : r134a, q138a and s136a directly or through a water molecule bridge.
it also interacts with the carbonyl oxygen of d113a.
in the c. albicans thiamine complex,  <dig> water molecules take the place of oxygen atoms from the proximal thiamine phosphate.
similarly to the s. cerevisiae apo form, a third water molecule occupies the same position as the distal thiamine phosphorus atom in the thiamine-pnp complex.
the side chain of the q <dig> can adopt two alternative conformations: the one observed in s. cerevisiae or mouse tpk  and the one observed uniquely in the c. albicans tpk/tpnp complex .
in the latter conformation, q <dig> does not interact with the inorganic phosphate described above.
magnesium binding site in the tpk/tpnp complex
three magnesium ions are stabilized in the active site.
the first two ions superimpose exactly with the ones co-crystallised in the c. albicans tpk structure with thiamine.
one is coordinated through the δ-oxygen of each of the four conserved aspartates and the oxygen of the proximal phosphate in tpnp.
the sixth mg2+ coordination involves a water molecule in c. albicans tpk instead of the amp-phosphoryl in the mouse structure .
the second one is stabilized by the same interactions as in the thiamine/tpk complex except for the o <dig> atom of the thiamine which is involved in a thiamine-pnp ester bond rather than an hydroxyl group in thiamine .
the third magnesium ion is coordinated by the δ-oxygen from d <dig> and d <dig> and one oxygen atom from each phosphate group of the thiamine-pnp.
the last coordination of this mg2+ involves an oxygen atom from the free inorganic phosphate symmetrically positioned with regard to the amp phosphate shown in the mouse atp binding site .
discussion
identification of a potential secondary binding site
timm et al.
described the 3d structure of the mouse thiamine pyrophosphokinase co-crystallized with amp, pyrithiamine pyrophosphate  and magnesium ions  <cit> .
their results confirmed that the amp  is maintained in the protein by interactions with residues  <dig>   <dig>   <dig> to  <dig>   <dig> to  <dig>   <dig> to  <dig>   <dig> and  <dig>  although we did not obtain any nucleotide analogue in our structures along with the tpnp and magnesium ions, we have identified an inorganic phosphate in the structure located differently to the amp molecule in the mouse tpk structure.
this phosphate is diametrically opposed to the amp phosphoryl group described by timm et al.
relative to the thiamine moiety  and can have two distinct origins.
first, amp-pnp is known to be very unstable in acidic conditions and can hydrolyze into an equivalent phosphoramidate and inorganic phosphate .
our crystals have been obtained at ph  <dig>  with a large excess of amp-pnp, but a partial degradation of amp-pnp cannot be excluded.
second, this phosphate may originate from the amp used in the pyrophosphorylation of the thiamine.
the co-crystallized inorganic phosphate highlights a very high symmetry in the active site .
the residues involved in this phosphate stabilization are poorly conserved across the different known tpks .
the stabilization of the non-phosphate part of the amp in this alternative site seems to be restricted to the ribose part of the nucleotide, which suggests the existence of a rather non specific binding site which could accommodate nucleotides other than atp, namely gtp, utp and ttp  <cit> .
alternatively, it could correspond to a transitional site for the amp on its path out of the protein, or for the atp on its way to the active site, and thus guide the motion of this product/substrate during the hydrolysis process.
existence of a gatekeeper residue?
mutational studies of the human tpk, sharing  <dig> % identity over  <dig> amino-acids with the c. albicans tpk, showed that residues d <dig>  d <dig> and d <dig>  play a crucial role in carrying out the catalytic process, that q <dig> and d <dig>  are involved in the binding of thiamine, and that t <dig> and r <dig>  interact with the atp  <cit> .
in our structures, q138a adopts two main conformations: one opened and one closed with respect to the active site access.
the structure containing thiamine only exhibits the closed conformation, while both are present in the tpk/tpnp complex.
as in other kinase structures, q138a could play the role of a gatekeeper, one of the major determinants of the selectivity in these proteins  <cit> .
if a secondary binding site exists in the protein, this residue could directly interact with the phosphoryl group of the amp and both the ribosyl and pyrimidine part of the nucleotide , thus playing a role in the enzyme selectivity.
in the event of a transitional site, q138a could modulate the release of amp or the entrance of atp.
hypothesis on the pyrophosphorylation mechanism in c. albicans tpk
the mechanism of pyrophosphorylation is not well understood.
structural studies complemented by enzymatic assays and mutation experiments, could give us insight into this process conserved in a lot of species.
baker et al.
<cit>  proposed that, in yeast tpk, the three aspartates corresponding to d78a, d113a, a142a could play the same role as in the hppk .
in this enzyme, the pyrophosphate  is transferred from an atp to an hp  through a one step pyrophosphorylation mechanism.
the ppi is maintained in the proper conformation by two aspartates through the coordination of two mg2+ ions  <cit> .
in an attempt to co-crystallize tpk with its two substrates or in an intermediate state, we used amp-pnp and amp-cpp, two atp analogues.
in the amp-pnp, the bond between β and γ phosphates is non-hydrolysable, excepted under acidic conditions, whereas in amp-cpp it is the bond between α and β phosphates which is non-hydrolysable.
our attempts to co-crystallize amp-cpp or amp-pnp with the enzyme have failed.
nevertheless, using amp-pnp, as suggested by the electronic density maps, the pnp group was transferred to the thiamine to form a thiamine-pnp.
this observation is consistent with a one step ppi transfer.
a steady-state kinetics study of the human tpk led to the proposal that the mechanism is a ping-pong reaction  <cit>  as proposed for yeast  <cit> .
according to our results, and in agreement with timm et al., the possibility to co-crystallize the two substrates at the same time is inconsistent with an ordered ping-pong mechanism where the thiamine binds the enzyme after atp has been hydrolyzed and the amp released.
moreover, the presence and position of a possible secondary amp binding site is more likely to support a mechanism where the amp is released after the ppi transfer and before the tpp molecule.
to our knowledge, this study is the first to report more than one magnesium ion in the tpk active site with one or two additional mg2+ properly positioned to participate in the pyrophosphorylation process in a mechanism akin to the one proposed by timm et al.
<cit> .
where timm et al.
showed a water molecule, our structures exhibit a magnesium ion at a strategic location.
after the binding of an atp molecule, k145a, a strictly conserved lysine, could activate the thiamine hydroxyl extremity through the coordination of the first mg2+.
at the same time, the  <dig> aspartates  could activate the ppi moiety of the atp molecule.
the transfer could then take place.
each c. albicans structure reveals new positions suggesting that  <dig> mg2+ ions could be involved in the ppi transfer mechanism.
the third mg2+ could stabilize the ppi during the transfer.
CONCLUSIONS
in summary, we have determined the x-ray structure of the c. albicans thiamine pyrophosphokinase co-crystallized with the thiamine or the thiamine-pnp and an inorganic phosphate.
these different structures allow us to describe several original features.
the active site symmetry and the inorganic phosphate binding site suggest the presence of an alternative binding site in c. albicans tpk, or at least a transitional position of the substrate/product along the reaction pathway.
this also suggests an enzymatic process where both products, tpp and amp, are present at the same time in the protein with the release of the amp molecule preceding the release of the tpp.
we propose that the q138a could interact with both substrates and products and work as a gatekeeper, modulating the activity of the enzyme .
the crystallization of thiamine-pnp at the tpk active site strongly suggests that the pyrophosphorylation process is a one step mechanism.
k145a seems to be important in the catalytic process and substrate activation.
the presence of two or three magnesium ions at the same time in the active site in these structures suggests a catalytic reaction similar to the one described for hppk, also performing pyrophosphate transfer.
the ppi transfer mechanism at work in thiamine pyrophosphokinases could be further explored by studies focusing on mimicking the transition state.
abbreviations
tpk: thiamine pyrophosphokinase; tpp: thiamine pyrophosphate; ppi: pyrophosphate; tpnp: thiamine-pnp; amp-pnp: adenosine 5'-triphosphate; pnp: diphosphate; hppk: 6-hydroxymethyl- <dig>  8-dihydropterin pyrophosphokinase; lic: ligation-independent cloning; ncs: non crystallographic symmetry.
authors' contributions
ss performed research, analyzed data and wrote the paper.
vm, nm and tr designed research and contributed to new reagents/analytic tools.
cs: performed research.
j–mc: designed research and wrote the paper.
ca designed research, performed research, analyzed data and wrote the paper.
all authors read and approved the final manuscript.
footnotes
the atomic coordinates and structure factors for the crystal structures of the thiamine pyrophosphokinase from candida albicans in complex with thiamine or thiamine-pnp are available in the rcsb protein data bank under pdb id 2hh <dig> and 2g9z respectively.
supplementary material
additional file 1
psf- <dig> expression vector map.
click here for file
additional file 2
stereo ribbon diagram of the c. albicans tpk monomer structure color coded according to secondary structure elements .
click here for file
additional file 3
molecular surface representation of the c. albicans tpk active site.
conserved residues in contact with at least one ligand are colored in blue and variable ones in red.
all other residues are colored in grey.
the ligands are colored with the pymol default colors and mg2+ ions are in white.
click here for file
additional file 4
graph representation of the b-factors values of the c. albicans tpk structures in complex with thiamine and thiamine-pnp.
monomers a and b of tpk/thiamine-pnp are shown in black and grey respectively.
monomers a and b of tpk/tiamine are shown in red and orange respectively.
click here for file
acknowledgements
the profun project was supported by a grant from the french ministry of research and education  and by centre national de la recherche scientifique .
we acknowledge the use of the marseille-nice génopole bioinformatic platform, the ibsm proteomic platform and the support of the experimental team of the igs laboratory.
we also want to thank the esrf teams on bm30a and id <dig> for helpful assistance and dr. pascal hingamp for reading of the manuscript.

 our laboratory runs a structural genomics project targeting fungal protein-coding genes in search of new anti-fungal targets. the main goal of this project is to quantitatively express the selected saccharomyces cerevisiae and candida albicans genes products to characterize them functionally and structurally as well as explore their potential as new drug targets. the genes selected in candida albicans are of two types: 1) they have orthologous genes in saccharomyces cerevisiae that are essential or 2) they are conserved in pathogenic fungal genomes and can be absent from the non-pathogenic yeast saccharomyces cerevisiae. candida albicans is a diploid organism with eight sets of homologous chromosomes and a genome size of about mb. this pathogen is part of the normal microflora of the human gastrointestinal tract or oropharynx and is responsible for various nosocomial infections, potentially lethal in immunocompromised patients or patients in the intensive care unit. the ca protein is encoded by a gene conserved across all known fungal genomes. it shares 36% sequence identity with saccharomyces cerevisiae yor143c, a thiamine pyrophosphokinase essential for fungus growth and survival. this enzyme is also essential in s. pombe. the production of thiamine pyrophosphate, catalyzed by tpk, is critical for both catabolic and anabolic cellular processes. in vertebrates, a deficiency in thiamine synthesis is linked to neurological diseases such as beriberi and wernicke encephalopathy. in all synthesizing organisms, thiamine is produced from phosphorylated het thiazole) and hmp that are condensed into thiamine phosphate. in yeast, this thiamine phosphate is then hydrolyzed into thiamine before pyrophosphorylation by the tpk that catalyzes the transfer of a pyrophosphate from atp to thiamine, resulting in amp and tpp. this article reports the enzymatic characterization as well as the structure determination of the candida albicans ca thiamine pyrophosphokinase in complex with both thiamine and thiamine-pnp. we also present a comparison of ca with the structure of the mouse tpk ternary complex with pyrithiamine pyrophosphate and amp, with the tpk s. cerevisiae structure and with sequences from other species. methods general cloning strategy the cdnas corresponding to the tpk genes were amplified by pcr from their respective genomic dnas. gene cloning was performed using the ligation-independent cloning method based on ligation of sticky ends generated by t dna polymerase. for the purpose of the profun project, we constructed a specific expression vector, psf- patent, compatible with lic cloning using the following procedure. the ecori+bamhi region of pqe was inserted in pqe80l vector where xhoi, ncoi and mfei sites have been mutated. then the lacz encoding gene was pcr amplified using primers containing flanking sequences used for lic cloning: 5'-ccatggctcatcaccatcaccatcacgggcatcaccatcaattg, forward primer containing the coding sequence for a poly-histidine tag, 5'-ggatccctcgagttagtcaccatccaattg, reverse primer. in the absence of an insert, the psf- expression vector expresses the lacz gene, while the in frame insertion of the resulting pcr products into ncoi+bamhi sites deactivates the gene thus allowing the easy detection of the parental vector. in addition; the gfp encoding gene was pcr-amplified using primers containing flanking sequences 5'-ctcgaggcggccg and 5'-ggatccattatgcggccgc. this pcr product was inserted at the 3'end of the lacz encoding gene into xhoi+bamhi sites yielding the psf- expression vector that expresses the gfp in c-terminal fusion with the targeted gene. not restriction sites flanking the gfp encoding gene allows its removal. cloning and expression screening of ca and yor143c tpk genes pcr amplifications were performed using primers specific for ca and yor143c tpk genes preceded by 5'-catcaccatcaattg and 5'-tcaccatccaattg together with purified candida albicans and saccharomyces cerevisiae genomic dna as template. the pcr products were directly purified using the nucleospin extract kit. then pmol of the purified pcr product was treated with t dna polymerase in the presence of mm of dctp for min. at 22°c before inactivating the enzyme by heating min at 75°c. in a parallel procedure, the expression vector, psf- was digested with the mfel restriction enzyme to excise the insert bearing the lacz encoding sequence. psf- was then purified on agarose gel using the nucleospin extract kit and treated with t dna polymerase in the presence of mm of dgtp for min at 22°c before inactivating the enzyme by heating min. at 75°c. the cloning step of the two fungi tpk genes consisted in a hybridization reaction performed by mixing pmol of psf- and pmol of the insert in a reaction volume of μl and an incubation time of min. at 22°c, μl of mm edta was then added to the reaction mixture. after a second incubation period of min. at 22°c, the entire hybridization reaction was used to transform e. coli dh5α. selection was performed on lb plates containing μg/ml ampicillin, positive plasmids were then isolated. this cloning procedure allowed insertion of the gene of interest in frame with a sequence encoding the n-terminal tag and a ghhhql sequence corresponding to the translation of the forward primer sequence. an additional c-terminal fragment qldgdleaa corresponds to the translation of a linker between the gene of interest and the sequence encoding the gfp. an expression screening was performed using our standard procedure and the gfp gene reporter was used to quantify the soluble expression through fluorescence measurements in order to identify the best condition for the fungi tpk proteins soluble expression. subsequent removal of the gfp encoding gene was achieved by digesting the plasmid by noti followed by intra ligation. as a result, the plasmids carrying the ca gene were over-expressed in e. coli origami in 1l flasks containing sb medium cultured overnight at 17°c. induction was performed using iptg when the culture reached an od nm of. the plasmids carrying the yor143c gene were expressed in e. coli origami in 1l flasks containing 2ytg medium at 25°c after induction with iptg when the culture reached an od nm of. after centrifugation, the pellets were resuspended in buffer a with % glycerol and % triton x- then sonicated and centrifuged again. purification the cleared lysate was applied to a ml hitrap chelating column loaded with ni2+ and equilibrated with buffer a. the column was washed with column volumes of buffer a, column volumes of buffer a containing mm imidazole and column volumes of buffer a containing mm imidazole at a flow rate of ml.min- elution was performed with a linear gradient over column volumes from mm to mm imidazole. the fractions corresponding to the elution of ca and yor tpk proteins with 150– mm imidazole were run on a desalting column and analyzed by mass spectroscopy and n-terminal edman sequencing. after purification, the fractions contained at least 98% pure protein in tris buffer mm ph and tris buffer mm, mm nacl ph. for the ca protein, the isoelectrofocalisation revealed a band around instead of the predicted pi of. gel filtration of the purified tpk proteins on a sephacryl s hr column indicated the final products were dimeric in solution. enzymatic assays in order to verify that our two c. albicans and s. cerevisiae enzymes were active tpk we developed a protocol involving four consecutive reactions and different enzymes. the tpk activities were determined at room temperature using purified protein in tris buffer in presence of mm thiamine and mm atp. the reaction cascade was monitored through the nadh decrease in absorbance at λ = nm. the first reaction is catalyzed by the tpk and produces a tpp and an amp. the myokinase then uses the produced amp in the presence of atp to produce adp molecules, each of them being used by the pyruvate kinase enzyme in presence of phosphoenol pyruvate to produce a pyruvate and an atp molecule. the last step is catalyzed by the lactate dehydrogenase hydrolyzing pyruvate in the presence of nadh to produce l-lactate and nad+. crystallisation the c. albicans tpk recombinant protein was concentrated to g/l in mm tris buffer at ph using a centrifugal filter device. precipitation experiments were carried out at k using various precipitating agents at various phs to determine the optimal protein concentration for crystallization. the screening for crystallization conditions was performed on × 96-well crystallization plates loaded by an 8-needle dispensing robot, using a μl sitting drop per condition at k. the tested crystallization conditions include in-house designed and commercially available solution sets. the tpk protein was then incubated with amp-pnp and/or thiamine for few minutes and the best crystals of each protein/ligand complex were obtained using the hanging drop vapor diffusion method with a ml reservoir. crystallization droplets were made of μl of complex mixed with μl of the reservoir solution made of to % peg mgcl m, tris m, % glycerol between ph and. crystals appeared within a few days. data collection crystals of the tpk protein were collected in a hampton research mm loop, flash frozen to k in a cold nitrogen gas stream and subjected to x-rays. a first data set was collected from a crystal of tpk incubated with thiamine on a marccd camera at the european synchrotron radiation facility on the bm30a-fip beamline at a wavelength of Å. a second data set corresponding to a crystal of tpk incubated both with thiamine and amp-pnp was collected at esrf on id beamline on an adsc q 2d detector at a wavelength of Å. the diffraction data were indexed with mosflm and scaled with the scala software from the ccp suite. the two crystals belong to the space group p with two molecules in the asymmetric unit. all statistics of the processed data are summarized in table values in parentheses are for the highest resolution shell. < i/σi>, is the mean signal to noise ratio, where i is the integrated intensity of a measured reflection and σ is the estimated error in the measurement. rsym = ∑h ∑i|ih, i - ⟨ih⟩|/∑h ∑i|ih, i, where i is the integrated intensity of reflection h having i observations and ⟨ih⟩ is the mean recorded intensity of reflection h over multiple recording. rcryst = ∑||fo|-|fc||/∑|fo|, where fo are observed and fc calculated structure factor amplitudes. rfree is calculated from a randomly chosen % of reflections. structure determination the c. albicans tpk crystal structure in complex with thiamine has been determined by molecular replacement using the caspr server. to generate the c. albicans tpk models, we used the available three-dimensional structures of saccharomyces cerevisiae and mus musculus thiamine pyrophosphokinase as template and tpk related sequences from other species. modeller produced models which were screened for a molecular replacement solution using to Å data and the amore software. the space group was first misinterpreted as c with one monomer per asymmetric unit and cell parameters of a = b = c =, β =. the structure was solved in this space group. the best model produced a solution with one monomer and a correlation of % and a r-factor of % prior to cns refinement. after one round of rigid-body refinement and minimization using the cns program with to Å data, the r-factor dropped to 42% with a 50% rfree. rapidly, the refinement stopped converging and we suspected wrong symmetry assignation. we thus reprocessed the tpk/thiamine/amp-pnp data in p space group and computed a self-rotation function using amore which resulted in a peak corresponding to a two-fold pseudo symmetry with a correlation coefficient of % using data between and Å thus confirming the p space group. the two monomers, related by a non crystallographic symmetry, mainly present differences in flexible loops and water molecules. the model was manually corrected using turbo-frodo followed by several rounds of minimization and individual b-factors refinements using to Å data and the cns software. water molecules were built into the model as well as a thiamine-pnp molecule, three mg2+ and one cl- ion per monomer. some residues were still disordered in loops l l and l and are absent from the deposited structure. the final free and working r-values are % and % respectively. the refined structure was then used against the tpk/thiamine data and refinement was performed using several manual rebuilding cycles followed by minimization and b-factor refinement using coot and refmac. water molecules were built into this model as well as a thiamine molecule and two mg2+ ions per monomer. again, some residues were still disordered in loops l l l and l and are absent from the deposited structure. the final free and working r-values are % and % respectively.  tpk activity of the c. albicans recombinant protein the activity of the s. cerevisiae tpk protein was previously measured on the native protein extracted from yeast. to validate our indirect enzymatic assay, we used the s. cerevisiae enzyme as a tpk activity control. under the conditions described in the methods section we measured specific activities of nm/min/mg for the s. cerevisiae enzyme and of nm/min/mg for the c. albicans recombinant protein. the ca overall structure: monomer as expected, the overall structure of the candida albicans tpk is similar to other members of the thiamine pyrophosphokinase family. ca is a homodimer, with each monomer constituted of two domains. the d domain is an αβ fold with a twisted β-sheet made of six parallel strands surrounded by six helices. the d domain is a β sandwich composed of two layers arranged in a jelly-roll topology. antiparallel strands β β β β 13-β β and β parallel to β constitute the first layer. antiparallel strands β β β and β constitute the second one. both domains contribute to the dimeric association with a Å buried surface area. we used the 3d-coffee server to perform a structural alignment of selected tpk sequences of various origins using ca saccharomyces cerevisiae and mus musculus tpk as structural templates. despite the high variability among these tpk sequences, there is strong residue conservation around the catalytic center. four aspartates are strictly conserved and have already been proposed to be involved in the stabilization of the atp molecule through mg2+ contacts. an aromatic residue is always found at position and is known to participate in the thiamine stabilization. an asparagine residue is strictly conserved but its function is not yet understood. this residue is close enough to the thiamine substrate to make some contacts with its amidopyrimidine ring. interestingly, there are three loops in the c. albicans tpk structure. two of them, l and l, are unique to c. albicans, while the third and longest insert is also present in the yeast homolog with which it shares less than 20% identity over residues. none of these fragments appear to be directly involved in the catalytic reaction or in the ligands binding. in the structure, the l loop lies in the solvent, in an open conformation and away from the rest of the molecule. the b-factors for this loop are also very high. we first built an approximate loop by following the residual density present in the 2fo-fc and fo-fc maps. we then used manual docking to verify that the length and geometry of this loop was compatible with a transient interaction with the ligands. this loop may thus play a role during the binding of the substrates, or in the pyrophosphorylation mechanism itself. the ca overall structure: dimer the main differences between the two monomers related by the ncs involve flexible loops disordered in the crystal structure. they include loops l l and l which are too distant to be involved in the active site formation and located too far from symmetry related molecules to possibly be directly involved in the crystal packing. there are also water molecules which are not strictly symmetrical between the two monomers. although the loop l is highly mobile and associated with high b-factors it presents little differences between the two monomers. the l8-α5-l fragment, which is involved in crystal packing through interactions between l8-α helix/turn and α3-β helix/turn in the two monomers, can be superposed with less than Å root mean square deviation based on α carbon superposition. there are no major differences between the tpk known structures based on cα superposition using lsqman. rmsd are Å between the c. albicans and the s. cerevisiae tpk structures, and Å between the c. albicans and the mouse tpk. the backbone trace around the thiamine binding site superimposes well except for one fragment. timm et al. described a rearrangement of the l loop in the mouse tpk when comparing the structures with thiamine and tpp. this rearrangement is not observed within the c. albicans tpk which fits the mouse tpk in complex with tpp except for the s side chain. thiamine binding site two binding sites can be shown in the asymmetric unit. the strong electronic density is consistent with thiamine molecules present at the thiamine binding site. as in the mouse structure, the thiamine moieties are located at an extremity of the groove formed by the dimer assembly. for convenience, we will take a as the reference monomer and b as the second one but all observations made on one of the active sites can be transposed to the second one. the ligands are maintained by both main chains and side chains interactions. these interactions involve the strands β β β of monomer b and the loops l l of monomer a. as observed in other structures there is a weak stacking between an aromatic residue and the amidopyrimidine ring. the cm carbon makes hydrophobic interactions with the w290b side chain, a residue conserved in the sequence of the s. cerevisiae homologue. in the mouse sequence this residue is replaced by an aspartate and does not make any contact with the ligand. nevertheless, a certain hydrophobic environment is maintained in the murine structure with the spatial proximity of a leucine. the thiamine ammonium group n of the amidopyrimidine ring is engaged in a salt bridge with the q138a main-chain carbonyl oxygen. the opposite side of this ring is stabilized by another salt bridge involving n and the oxygen from the s299b side chain. the aspartate residue interacting with n and n described in the mouse structures is replaced by a tyrosine too far away from the ring to interact with it. n from the thiazolium ring is stabilized by electrostatic interactions between the main chain carbonyl oxygen atoms of s300b and q138a. at least residues involved in the thiamine binding pocket are poorly conserved between different species but well represented in fungus and could be the focus of specific antifungal drug design. magnesium binding site in the tpk/thiamine complex as observed in the mouse tpk/tpp complex, an mg2+ ion is maintained by electrostatic interactions with the side chain of the four strictly conserved aspartate residues. the last coordinations are made with water molecules and. timm et al. highlighted that a second mg2+ ion could be involved in the reaction process as in another pyrophosphokinase. interestingly, the c. albicans tpk/thiamine complex reveals a second mg2+ partially stabilized by the hydroxyl group o of thiamine and a salt bridge with d and d side chains and with two main chain carboxy oxygens. the nitrogen atom from the k side chain participates in the last coordination. as proposed by timm et al., this lysine could be involved in the activation of the thiamine hydroxyl by the stabilization of a water molecule or, as in this c. albicans structure, of a magnesium ion. thiamine-pnp binding site, implications for the atp binding site in order to compare the c. albicans and mouse tpk binding sites, we used atp analogues during the crystallization process. in the amp-cpp molecule a carbon atom replaces the oxygen atom located between the α and β phosphates in the atp molecule. in the amp-pnp molecule, a nitrogen atom replaces o while we did not get crystals of the tpk/thiamine/amp-cpp complex, the tpk incubated with thiamine and amp-pnp produced usable crystals. however, the structure revealed that instead of a tpk/thiamine/amp-pnp complex, we obtained an unexpected tpk/thiamine-pnp complex with a tpnp molecule in the enzyme binding site. this result is consistent with the earlier suggestion that the pyrophosphate group is transferred at once from the atp molecule to the thiamine moiety. the superimposition of the c. albicans tpk/tpnp complex onto the mouse tpk/tpp complex highlights few differences between the two thiamine phosphate ends. the nitrogen n in the tpnp molecule is symmetrically opposed to its equivalent oxygen o in the tpp molecule. it is stabilized by a weak electrostatic interaction with the s301b γ-oxygen in the c. albicans tpk/tpnp complex. in the mouse tpk/tpp complex, this serine residue, located in the amp binding pocket, is at Å from the o we propose that this serine residue could repulse the tpp in normal conditions and help to release it out of the active site. in the mouse tpk/tpp complex, q134a interacts directly with the phosphate end of the thiamine. the equivalent glutamine adopts the same conformation in the c. albicans tpk/thiamine complex and an opened conformation in the c. albicans tpk/tpnp complex. in the mouse tpk complex with amp and tpp, the atp binding site involves residues poorly conserved across different species. in the c. albicans tpk structure, no extra density that could correspond to a nucleotide was found even if this site is opened enough to accommodate an atp molecule. interestingly, exactly opposed to the mouse amp binding site,, we have identified an inorganic phosphate. this phosphate is stabilized by a glutamine, an arginine and a salt bridge involving a magnesium ion. moreover, despite the lack of strong residual density, an amp molecule could reasonably be accommodated at that position with an average b-factor of and a good occupancy. however, the non-phosphate part of this putative amp molecule presents higher b-factors and interacts with side chains of poorly conserved residues: r134a, q138a and s136a directly or through a water molecule bridge. it also interacts with the carbonyl oxygen of d113a. in the c. albicans thiamine complex, water molecules take the place of oxygen atoms from the proximal thiamine phosphate. similarly to the s. cerevisiae apo form, a third water molecule occupies the same position as the distal thiamine phosphorus atom in the thiamine-pnp complex. the side chain of the q can adopt two alternative conformations: the one observed in s. cerevisiae or mouse tpk and the one observed uniquely in the c. albicans tpk/tpnp complex. in the latter conformation, q does not interact with the inorganic phosphate described above. magnesium binding site in the tpk/tpnp complex three magnesium ions are stabilized in the active site. the first two ions superimpose exactly with the ones co-crystallised in the c. albicans tpk structure with thiamine. one is coordinated through the δ-oxygen of each of the four conserved aspartates and the oxygen of the proximal phosphate in tpnp. the sixth mg2+ coordination involves a water molecule in c. albicans tpk instead of the amp-phosphoryl in the mouse structure. the second one is stabilized by the same interactions as in the thiamine/tpk complex except for the o atom of the thiamine which is involved in a thiamine-pnp ester bond rather than an hydroxyl group in thiamine. the third magnesium ion is coordinated by the δ-oxygen from d and d and one oxygen atom from each phosphate group of the thiamine-pnp. the last coordination of this mg2+ involves an oxygen atom from the free inorganic phosphate symmetrically positioned with regard to the amp phosphate shown in the mouse atp binding site. discussion identification of a potential secondary binding site timm et al. described the 3d structure of the mouse thiamine pyrophosphokinase co-crystallized with amp, pyrithiamine pyrophosphate and magnesium ions. their results confirmed that the amp is maintained in the protein by interactions with residues to to to and although we did not obtain any nucleotide analogue in our structures along with the tpnp and magnesium ions, we have identified an inorganic phosphate in the structure located differently to the amp molecule in the mouse tpk structure. this phosphate is diametrically opposed to the amp phosphoryl group described by timm et al. relative to the thiamine moiety and can have two distinct origins. first, amp-pnp is known to be very unstable in acidic conditions and can hydrolyze into an equivalent phosphoramidate and inorganic phosphate. our crystals have been obtained at ph with a large excess of amp-pnp, but a partial degradation of amp-pnp cannot be excluded. second, this phosphate may originate from the amp used in the pyrophosphorylation of the thiamine. the co-crystallized inorganic phosphate highlights a very high symmetry in the active site. the residues involved in this phosphate stabilization are poorly conserved across the different known tpks. the stabilization of the non-phosphate part of the amp in this alternative site seems to be restricted to the ribose part of the nucleotide, which suggests the existence of a rather non specific binding site which could accommodate nucleotides other than atp, namely gtp, utp and ttp. alternatively, it could correspond to a transitional site for the amp on its path out of the protein, or for the atp on its way to the active site, and thus guide the motion of this product/substrate during the hydrolysis process. existence of a gatekeeper residue? mutational studies of the human tpk, sharing % identity over amino-acids with the c. albicans tpk, showed that residues d d and d play a crucial role in carrying out the catalytic process, that q and d are involved in the binding of thiamine, and that t and r interact with the atp. in our structures, q138a adopts two main conformations: one opened and one closed with respect to the active site access. the structure containing thiamine only exhibits the closed conformation, while both are present in the tpk/tpnp complex. as in other kinase structures, q138a could play the role of a gatekeeper, one of the major determinants of the selectivity in these proteins. if a secondary binding site exists in the protein, this residue could directly interact with the phosphoryl group of the amp and both the ribosyl and pyrimidine part of the nucleotide, thus playing a role in the enzyme selectivity. in the event of a transitional site, q138a could modulate the release of amp or the entrance of atp. hypothesis on the pyrophosphorylation mechanism in c. albicans tpk the mechanism of pyrophosphorylation is not well understood. structural studies complemented by enzymatic assays and mutation experiments, could give us insight into this process conserved in a lot of species. baker et al. proposed that, in yeast tpk, the three aspartates corresponding to d78a, d113a, a142a could play the same role as in the hppk. in this enzyme, the pyrophosphate is transferred from an atp to an hp through a one step pyrophosphorylation mechanism. the ppi is maintained in the proper conformation by two aspartates through the coordination of two mg2+ ions. in an attempt to co-crystallize tpk with its two substrates or in an intermediate state, we used amp-pnp and amp-cpp, two atp analogues. in the amp-pnp, the bond between β and γ phosphates is non-hydrolysable, excepted under acidic conditions, whereas in amp-cpp it is the bond between α and β phosphates which is non-hydrolysable. our attempts to co-crystallize amp-cpp or amp-pnp with the enzyme have failed. nevertheless, using amp-pnp, as suggested by the electronic density maps, the pnp group was transferred to the thiamine to form a thiamine-pnp. this observation is consistent with a one step ppi transfer. a steady-state kinetics study of the human tpk led to the proposal that the mechanism is a ping-pong reaction as proposed for yeast. according to our results, and in agreement with timm et al., the possibility to co-crystallize the two substrates at the same time is inconsistent with an ordered ping-pong mechanism where the thiamine binds the enzyme after atp has been hydrolyzed and the amp released. moreover, the presence and position of a possible secondary amp binding site is more likely to support a mechanism where the amp is released after the ppi transfer and before the tpp molecule. to our knowledge, this study is the first to report more than one magnesium ion in the tpk active site with one or two additional mg2+ properly positioned to participate in the pyrophosphorylation process in a mechanism akin to the one proposed by timm et al.. where timm et al. showed a water molecule, our structures exhibit a magnesium ion at a strategic location. after the binding of an atp molecule, k145a, a strictly conserved lysine, could activate the thiamine hydroxyl extremity through the coordination of the first mg2+. at the same time, the aspartates could activate the ppi moiety of the atp molecule. the transfer could then take place. each c. albicans structure reveals new positions suggesting that mg2+ ions could be involved in the ppi transfer mechanism. the third mg2+ could stabilize the ppi during the transfer.  in summary, we have determined the x-ray structure of the c. albicans thiamine pyrophosphokinase co-crystallized with the thiamine or the thiamine-pnp and an inorganic phosphate. these different structures allow us to describe several original features. the active site symmetry and the inorganic phosphate binding site suggest the presence of an alternative binding site in c. albicans tpk, or at least a transitional position of the substrate/product along the reaction pathway. this also suggests an enzymatic process where both products, tpp and amp, are present at the same time in the protein with the release of the amp molecule preceding the release of the tpp. we propose that the q138a could interact with both substrates and products and work as a gatekeeper, modulating the activity of the enzyme. the crystallization of thiamine-pnp at the tpk active site strongly suggests that the pyrophosphorylation process is a one step mechanism. k145a seems to be important in the catalytic process and substrate activation. the presence of two or three magnesium ions at the same time in the active site in these structures suggests a catalytic reaction similar to the one described for hppk, also performing pyrophosphate transfer. the ppi transfer mechanism at work in thiamine pyrophosphokinases could be further explored by studies focusing on mimicking the transition state. abbreviations tpk: thiamine pyrophosphokinase; tpp: thiamine pyrophosphate; ppi: pyrophosphate; tpnp: thiamine-pnp; amp-pnp: adenosine 5'-triphosphate; pnp: diphosphate; hppk: 6-hydroxymethyl- 8-dihydropterin pyrophosphokinase; lic: ligation-independent cloning; ncs: non crystallographic symmetry. authors' contributions ss performed research, analyzed data and wrote the paper. vm, nm and tr designed research and contributed to new reagents/analytic tools. cs: performed research. j–mc: designed research and wrote the paper. ca designed research, performed research, analyzed data and wrote the paper. all authors read and approved the final manuscript. footnotes the atomic coordinates and structure factors for the crystal structures of the thiamine pyrophosphokinase from candida albicans in complex with thiamine or thiamine-pnp are available in the rcsb protein data bank under pdb id 2hh and 2g9z respectively. supplementary material additional file 1 psf- expression vector map. click here for file additional file 2 stereo ribbon diagram of the c. albicans tpk monomer structure color coded according to secondary structure elements. click here for file additional file 3 molecular surface representation of the c. albicans tpk active site. conserved residues in contact with at least one ligand are colored in blue and variable ones in red. all other residues are colored in grey. the ligands are colored with the pymol default colors and mg2+ ions are in white. click here for file additional file 4 graph representation of the b-factors values of the c. albicans tpk structures in complex with thiamine and thiamine-pnp. monomers a and b of tpk/thiamine-pnp are shown in black and grey respectively. monomers a and b of tpk/tiamine are shown in red and orange respectively. click here for file acknowledgements the profun project was supported by a grant from the french ministry of research and education and by centre national de la recherche scientifique. we acknowledge the use of the marseille-nice génopole bioinformatic platform, the ibsm proteomic platform and the support of the experimental team of the igs laboratory. we also want to thank the esrf teams on bm30a and id for helpful assistance and dr. pascal hingamp for reading of the manuscript.
in search of new antifungal targets of potential interest for pharmaceutical companies, we initiated a comparative genomics study to identify the most promising protein-coding genes in fungal genomes. we report the enzymatic characterization and the crystallographic structure of the candida albicans thiamine pyrophosphokinase. the presence of an inorganic phosphate in the crystallographic structure opposite the known amp binding site relative to the thiamine moiety suggests that a second amp molecule could be accommodated in the c. albicans structure. since thiamine pyrophosphate is an essential product involved in a variety of metabolic pathways, proteins responsible for its production satisfied these two criteria. finally, the tpk/thiamine-pnp complex is consistent with a one step mechanism of pyrophosphorylation. a second criterion was that the corresponding gene in saccharomyces cerevisiae should be essential.
 in search of new antifungal targets of potential interest for pharmaceutical companies, we initiated a comparative genomics study to identify the most promising protein-coding genes in fungal genomes. one criterion was the protein sequence conservation between reference pathogenic genomes. a second criterion was that the corresponding gene in saccharomyces cerevisiae should be essential. since thiamine pyrophosphate is an essential product involved in a variety of metabolic pathways, proteins responsible for its production satisfied these two criteria.  we report the enzymatic characterization and the crystallographic structure of the candida albicans thiamine pyrophosphokinase. the protein was co-crystallized with thiamine or thiamine-pnp.  the presence of an inorganic phosphate in the crystallographic structure opposite the known amp binding site relative to the thiamine moiety suggests that a second amp molecule could be accommodated in the c. albicans structure. together with the crystallographic structures of the enzyme/substrate complexes this suggests the existence of a secondary, less specific, nucleotide binding site in the candida albicans thiamine pyrophosphokinase which could transiently serve during the release or the binding of atp. the structures also highlight a conserved glutamine residue which could interact with the atp α-phosphate and act as gatekeeper. finally, the tpk/thiamine-pnp complex is consistent with a one step mechanism of pyrophosphorylation. 