The initial characterization demonstrated that Uu-TK also has similar enzyme kinetic properties to hTK1, with specificity for pyrimidine deoxynucleosides and with dTTP serving as a feedba
Trang 1from Ureaplasma urealyticum and investigations of
possible drug targets for the enzyme
Urszula Kosinska1*, Cecilia Carnrot2*, Staffan Eriksson2, Liya Wang2 and Hans Eklund1
1 Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden
2 Department of Molecular Biosciences, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden
Two potential nucleoside kinase genes coding for a
thymidine kinase (TK) (EC 2.7.1.21) and a
deoxyadeno-sine kinase (EC 2.7.1.74) are found in all sequenced
Mollicute genomes [1–9] Deoxyadenosine kinase from
Mycoplasma mycoides ssp mycoides and TK from
Ureaplasma urealyticum (Uu-TK) have previously been
cloned and characterized [10,11] U urealyticum (also
called Ureaplasma parvum) is a human pathogen
colon-izing the urogenital tract and it is associated with
several pregnancy complications, e.g infertility, altered
sperm motility, chorioamnionitis and pneumonia in the
neonate [12]
Bacterial TKs show moderate sequence identity with human TK1 (hTK1) and mollicute TKs, e.g Uu-TK shares 29% sequence identity with hTK1 The initial characterization demonstrated that Uu-TK also has similar enzyme kinetic properties to hTK1, with specificity for pyrimidine deoxynucleosides and with dTTP serving as a feedback inhibitor [11,13] Uu-TK is less fastidious than hTK1 with regard to phosphate donors, using all nucleoside triphosphates with similar efficiency [11,13] No genes encoding the enzymes for the de novo pathway of deoxynucleotide biosynthesis have been found in U urealyticum, strongly suggesting
Keywords:
bacterial; crystallography; deoxythymidine;
nucleoside analogues; thymidine kinase
Correspondence
H Eklund, Department of Molecular
Biology, Swedish University of Agricultural
Sciences, PO Box 590, Biomedical Centre,
S-751 24 Uppsala, Sweden
Fax: +46 18 53 69 71
Tel: +46 18 4754559
E-mail: hasse@xray.bmc.uu.se
*Note
These authors contributed equally to this
work.
(Received 22 August 2005, revised 14
October 2005, accepted 21 October 2005)
doi:10.1111/j.1742-4658.2005.05030.x
Thymidine kinases have been found in most organisms, from viruses and bacteria to mammals Ureaplasma urealyticum (parvum), which belongs to the class of cell-wall-lacking Mollicutes, has no de novo synthesis of DNA precursors and therefore has to rely on the salvage pathway Thus, thymi-dine kinase (Uu-TK) is the key enzyme in dTTP synthesis Recently the 3D structure of Uu-TK was determined in a feedback inhibitor complex, dem-onstrating that a lasso-like loop binds the thymidine moiety of the feed-back inhibitor by hydrogen bonding to main-chain atoms Here the structure with the substrate deoxythymidine is presented The substrate binds similarly to the deoxythymidine part of the feedback inhibitor, and the lasso-like loop binds the base and deoxyribose moieties as in the com-plex determined previously The catalytic base, Glu97, has a different posi-tion in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5¢-OH of the substrate to form a hydrogen bond The phosphorylation of and inhibition by several nucleo-side analogues were investigated and are discussed in the light of the sub-strate binding pocket, in comparison with human TK1 Kinetic differences between Uu-TK and human TK1 were observed that may be explained by structural differences The tight interaction with the substrate allows minor substitutions at the 3 and 5 positions of the base, only fluorine substitu-tions at the 2¢-Ara position, but larger substitusubstitu-tions at the 3¢ position of the deoxyribose
Abbreviations
AZMT, 3¢-azido-methyl-dT; Ca-TK, Clostridium acetobutylicum thymidine kinase; dNK, deoxynucleoside kinase; FCPU, 3¢-fluoro-5-cyclopropyl-dU; FLT, 3¢-fluoro-dT; hTK, human thymidine kinase; TK, thymidine kinase; Uu-TK, Ureaplasma urealyticum thymidine kinase.
Trang 2that it has to rely solely on the salvage pathway for
synthesis of DNA precursors Thus, Uu-TK is a
gate-way for the biosynthesis of dTTP, suggesting that
Uu-TK is a good target for drug development
Recently the 3D structures of Uu-TK and cytosolic
hTK1 were determined [14,15] The structure of these
thymidine kinases differs significantly from the earlier
known deoxyribonucleoside kinases and form a
separ-ate structural family Humans carry four
deoxyribonu-cleoside kinases: cytosolic TK1 and deoxycytidine
kinase, and mitochondrial TK2 and deoxyguanosine
kinase Deoxycytidine kinase, TK2, and
deoxyguano-sine kinase form a homologous deoxynucleoside kinase
(dNK) family with similar structures This family also
includes the herpes viral TKs and the insect
multisub-strate deoxyribonucleoside kinase showing high activity
with dT [13] The phosphate donor binds in both
fam-ilies to an a⁄ b domain, but this domain in the TK1
family is more similar to other ATP-binding structures
in the RecA family [14] Furthermore, instead of the
helical part forming the substrate site and the LID
region being involved in binding of the phosphate
donor in the dNK family, there is a domain in hTK1
and Uu-TK that contains a structural zinc atom and
lasso-like loop In the feedback inhibitor complexes of
Uu-TK and hTK1, the lasso-like loop binds the
thymi-dine moiety of the feedback inhibitor by hydrogen
bonding by main-chain atoms [14] The structure of the
bacterial TK from Clostridium acetobutylicum (Ca-TK)
is very similar to the Uu-TK, but, in the absence of
substrate or feedback inhibitor, the substrate site is
open and the lasso-like loop is disordered [16]
In the absence of a true substrate complex for any
member of the TK1 family, we have now determined
the first structure of a member of the TK1 family in
complex with the substrate dT The possibilities for
drug design have been investigated by enzyme kinetics
and analyzed in view of substrate binding It appears
that a combination of substitutions at several positions
of the nucleoside can pick up the small differences
between mycoplasmic and human TK1, which suggests
the route for further advances
Results and discussion
Overall structure
The overall tetrameric structure of Uu-TK in the
sub-strate complex is very similar to that in the complex
with the feedback inhibitor dTTP Each subunit can
be superimposed, with rmsds for Ca atoms of
0.3–0.6 A˚ The main differences are located close to
the phosphate binding sites, where a flexible loop
conformation differs among the four subunits of the tetramer and among subunits in the two complexes (Fig 1A) In the present substrate complex structure, only subunit A in the tetramer has a completely visible loop, whereas in the dTTP complex, only subunits B and D have visible loops [14] In all other cytosolic
TK structures determined so far, this loop is visible in only a few subunits For example, in one of the inde-pendent structure determinations of hTK1, this loop is fully visible in one of eight subunits in the asymmetric unit [15] It may be that this loop is involved in phos-phate donor interactions, but no such complex has so far been determined In the Ca-TK structure, the prod-uct ADP is bound at the phosphate donor site In spite
of this, only part of the loop is ordered in one of the subunits [16] This conformation is similar to that observed in the Uu-TK–dTTP complex The phosphate donor site in the present Uu-TK–dT structure is occu-pied by water molecules and, in one subunit, a Tris molecule Although the enzyme was crystallized in the presence of the ATP analogue adenosine 5¢-[b,c-methy-lene]-triphosphate (p[CH2]ppA; AMP-PCP), there was
no density for this molecule
Substrate binding The substrate dT binds to the enzyme in a similar way
to the dT moiety of the feedback inhibitor dTTP (Figs 1B and 2) and has the same interactions with the main chain of the lasso-like loop This loop has the same conformation in both complexes in contrast to Ca-TK where no substrate was present in the crystals There, the lasso-like loop was disordered [16] The sub-strate site is obviously induced by subsub-strate binding The base is hydrogen-bonded to main-chain atoms: O2
to N in residue 180, N3 to O in residue 178, and O4
to N in residue 128 (Fig 2) The methyl group is posi-tioned in a hydrophobic pocket lined by Cb of Ser163,
Sd in Met21, and Cd1 in Leu124 The closest polar atom is the carbonyl oxygen of residue 126 O3¢ of the deoxyribose is hydrogen-bonded to the main-chain amino group of Gly182
Glu97 has different conformations in the substrate complex and the previously determined inhibitor com-plex In the dT complex, O5¢ is hydrogen bonded to Glu97, in good agreement with its role as the catalytic base for the phosphoryl transfer reaction (Fig 1B) In the inhibitor complex, the phosphates of the feedback inhibitor repel the glutamate side chain A shift in the catalytic base was observed when the substrate and feedback inhibitor complex of Drosophila dNK were compared, but the conformational change was larger
in that case [17] In Uu-TK, only the side chain
Trang 3chan-ges conformation, whereas in Drosophila dNK the shift
is accompanied by main-chain movements
Substrate specificity, nucleoside analogues With radiolabelled ATP and a fixed concentration (100 lm) of various nucleoside analogues, Uu-TK activity was measured by a TLC assay Table 1 shows that 5-halogenated analogues are good substrates and have the highest activities The iodo atom has the same van der Waals radius as the methyl group of thymine, and the corresponding analogue has an activity com-parable to that of dT The analogue with the smaller fluorine in the 5 position has a lower activity, slightly lower than that of dU, which has a hydrogen atom in the 5 position The chlorine substitution is an outlier
in the halogen substitution series, as it has the highest activity of the halogenated analogues There is no cor-relation between the electronegativity of the substituent and its activity in the phosphorylation reaction From these investigations, it appears that substitu-tions at the 5 position as large as a cyclopropyl group are tolerated, and for an ethyl group the activity is decreased to about half of that of dT Larger
substitu-Fig 2 Interactions between thymidine and Uu-TK Hydrogen bonds are shown as dotted lines The tight binding site for the 2¢ position between the main chain of Lys180-Ile181 and Met21 Any substitu-tion at the 2¢ posisubstitu-tion hinders proper closure of the lasso, and thereby weakens substrate co-ordination.
A
B
Fig 1 (A) The structure of one subunit of Uu-TK (yellow) with
con-formations of the flexible loop as found in Uu-TK in complex with
dT in subunit A (orange, A), Uu-TK in complex with dTTP (green, B)
and in hTK1 in complex with dTTP (grey, C) The conformation of
the loop shown in green (B) is very similar to that found in Ca-TK in
complex with ADP as well as the loops in chains C and D of the
Uu-TK–dT structure (B) Superimposition of the nucleotide-binding
region in the substrate complex (orange) and the inhibitor complex
(olive) The side chain of the catalytic Glu97 has different positions
in the two complexes In the substrate complex, it is in a
catalyti-cally favourable position pointing inwards the active site In the
inhibitor complex, the side chain is repelled by the phosphates of
the inhibitor.
Trang 4tions clash sterically with neighbouring residues in the
5 position binding pocket (Fig 2) With bulkier
modi-fications, the activity decreased and no activity was
seen with, e.g 5-(2-bromovinyl)-dU (data not shown),
which is also the case with hTK1 [18,19] This steric
hindrance in the 5 position correlates well with the
small hydrophobic binding pocket in the enzyme
Analogues with modifications in the N3 position
showed much lower activity than dT The compounds
had one to three carbons added in different
configura-tions and gave 15–20% activity (Table 1) The
hydro-gen-bonding between the N3 nitrogen in dT and the
main-chain carbonyl of residue 178 is lost with alkyl
substitutions in the N3 analogues tested A substitution
in this position will probably hinder the tight spacing of
the lasso-like loop and disturb proper binding
The 3¢-OH is in an exposed position that can tolerate
large substitutions (Table 1) The 3¢-OH of the
deoxy-ribose forms a hydrogen bond with the main-chain
amino group of Gly182 that is lost when the hydroxy
group is replaced Still, an electronegative substituent,
such as fluorine, retains 50% of the activity For the
analogue with no hydroxy group in the 3¢ position,
2¢,3¢-didehydro-T, the activity was 3% of that with
dT [11] 3¢-Modified analogues that contain polar groups, e.g 3¢-fluoro-dT (FLT) and 3¢-azido-dT (AZT) can still form a hydrogen bond with the amino group
of Gly182, whereas analogues with nonpolar atoms bound to the 3¢-carbon, such as 3¢-fluoro-methyl-dT (FMT) and 3¢-azido-methyl-dT (AZMT), cannot form any hydrogen bond The latter two showed 4–7-fold lower activity than with dT (Table 1) The correspond-ing a form of FMT and AZMT were inactive (data not shown) Such substitutions would clash with Met21 Analogues with modifications at both the 3¢ and 5 position had lower activity than their corresponding analogue with only one modification, e.g 3¢-fluoro-5-fluoro-dU (FFU) vs FLT This is probably also the case with analogues modified at both the 3¢ and 3N positions
Analogues with modifications at the 2¢ position showed the lowest activity (< 2%) of all analogues tested, e.g arabinosyl-dT, difluoro-dU and 2¢-chloro-dU (data not shown) This agrees well with the tight binding site that is crowded on both sides of the 2¢ position (Fig 2) The OH group in arabinosyl-dT would interact sterically with Tyr187, which is one of the important residues that keep the lasso in place The smaller fluorine in the 2¢-Ara position was accepted and fluoro-arabinosyl-5-iodo-dU (FIAU) and 2¢-fluoro-arabinosyl-5-methyl-dU (FMAU) showed 44% and 34% activity, respectively Any substitution on the other side, such as 2¢-difluoro-dU and 2¢-chloro-dU, would interact with the main-chain carbonyl of residue
180 (Fig 2)
Analogues as inhibitors Some analogues were chosen for further analysis as inhibitors of dT phosphorylation The IC50 values are presented in Table 2 dU, 5-fluro-dU (FdU) and
Table 1 Phosphorylation of nucleoside analogues by Uu-TK and
hTK1 The values are from one experiment repeated with similar
results (< 20% variation) The specific activity with dT was set to
100% (1900 units), and 100 l M [c- 32 P]ATP was used.
Substrate (100 l M )
Activity (%)
Reference Uu-TK hTK1
5-Fluoro-dU (FdU)a
5-Ethyl-dU (EtdU) b
61 50 95 80 [11,19], [19]
dU a
3-Methyl-dT (MeT)c
46 21 77 43 [11,19], [19]
3¢-Azido-dT (AZT) a
3¢-Azido-methyl-dT (AZMT) b
35 8 52 15 [11,19], [29]
2¢-Fluoro-arabinosyl-5-iodo-dU (FIAU) d
2¢-Fluoro-arabinosyl-5-methyl-dU
(FMAU) d
3¢-Fluoro-5-cyclopropyl-dU (FCPU) b
3¢-Fluoro-5-ethynyl-dU (FEU) b
Source of the compounds: aSigma-Aldrich; bN G Johansson,
Medivir, Stockholm, Sweden; c W Tjarks, College of Pharmacy, The
Ohio State University, Columbus, Ohio; d J Fox, Memorial Sloan
Kettering Cancer Institute, New York.
Table 2 IC50 values of selected nucleoside analogues with Uu-TK and hTK1 Substrate concentrations were 1 l M dT and 2 m M ATP for Uu-TK and 0.2 l M dT and 2 m M ATP for hTK1.
Substrate
IC50(l M )
Trang 53-methyl-dT (MeT) showed low ability to inhibit
Uu-TK, with IC50 values of 200–500 lm, using 1 lm
[3H]dT as substrate 5-Ethyl-dU (EtdU) had an
inter-mediate IC50 value (47 lm), whereas 2¢ and 3¢
ana-logues containing fluorine or azido substitutions were
relatively efficient inhibitors (IC50 values < 20 lm),
with 3¢-azido-5-iodo-dU (AZIU) having the lowest
IC50 value (6 lm) (Table 2) IC50 values for hTK1,
using 0.2 lm [3H]dT as substrate, were also determined
with the above analogues (Table 2) A lower dT
con-centration was used for hTK1 to compensate for the
lower Km value observed with hTK1 and dT (in the
presence of 2 mm ATP) [11,20] The results with hTK1
showed a pattern, with MeT, FIAU and AZMT
form-ing a group of quite poor inhibitors with IC50 values
of 60–100 lm The other analogues tested with hTK1
fell into a group with relatively high capacity to
inhi-bit, and their IC50 values ranged from 15 lm down to
below 1 lm for 3¢-azido-5-iodo-dU (AZIU), the best
inhibitor (Table 2)
FLT and 3¢-fluoro-5-cyclopropyl-dU (FCPU), with
IC50 values of 14 and 11 lm, respectively, were the
only analogues that showed higher activity with
Uu-TK than with hTK1 (Table 1) Kinetic studies were
performed with Uu-TK and hTK1 using FLT and
FCPU as variable substrates together with 0.5 mm
[c-32P]ATP These analogues had about 3.5–4-fold
higher kcatvalues than dT, but at the same time
five-fold higher Kmvalues, resulting in a slightly lower
effi-ciency than dT (Table 3) In the case of hTK1, FLT
showed in this experiment higher efficiency than FCPU
and dT
Comparison with hTK1
Overall, the relative phosphorylation rates of the
ana-logues tested with Uu-TK are equal to or lower than
the corresponding rates with hTK1 The exceptions are
FCPU and FLT, which show higher relative activity
with Uu-TK than with hTK1 FCPU has a cyclopropyl
substitution in the 5¢ position of the base, and its
bet-ter activity with Uu-TK is probably due to the slightly
larger binding pocket of Uu-TK This enzyme has a serine at position 163 where hTK1 has a threonine, and the extra methyl group makes the site narrower FCPU and FLT also have fairly low IC50 values, which should be an advantage for a potential inhibitor However, the corresponding IC50 values with hTK1 were still lower, and this was the case with seven out
of the nine nucleosides tested as inhibitors of dT phos-phorylation Only FIAU and AZMT had higher IC50 values with hTK1 than with Uu-TK Still, they were not very efficiently phosphorylated by Uu-TK
These results show that the capacity of a nucleoside
to be a good inhibitor does not directly correlate with its rate of phosphorylation Despite the high struc-tural similarities between Uu-TK and hTK1, the latter
is much easier to inhibit, as shown by its lower IC50 values Furthermore, these two enzymes show a 10-fold difference in Km values for dU and dU ana-logues [11,21] The reasons for these differences in function may be related to the differences around the
5 position of the substrate, where Uu-TK has a serine and hTK1 has the more hydrophobic threonine The analogues investigated so far may not be direct lead compounds for the further development
of selective Uu-TK inhibitors, but this study demon-strates the necessity to use both a structural and functional approach for identification of new inhibi-tors and alternative substrates when nucleoside
kinas-es are the targets Such inhibitors would be desirable, as they could serve as efficient antibiotics because U urealyticum lacks the capacity to synthes-ize DNA precursors de novo The most promising route for further drug development for Uu-TK from this study seems to be to explore further substitu-tions at the 5 position and 3¢ position and combina-tions thereof
Experimental procedures
Materials The radiolabelled substances [3H]dT (25 CiÆmmol)1) and [c-32P]ATP ( 3000 CiÆmmol)1) were purchased from Amersham Biosciences (Uppsala, Sweden) Recombinant Uu-TK and hTK1 were prepared as previously described [11,22]
Enzyme assay
TK activity was determined by using a DE-81 filter paper technique with [3H]Thd or by a phosphoryl-transfer assay with [c-32P]ATP, as previously described [11] The standard reaction mixture contained 50 mm Tris⁄ HCl, pH 7.6, 2 mm
Table 3 Kinetic parameters of FLT, FCPU and dT with Uu-TK and
hTK1 A fixed [c- 32 P]ATP concentration (0.5 m M ) was used kcat
values were calculated based on a subunit molecular mass of
27.5 kDa (Uu-TK) and 25.5 kDa (hTK1), respectively.
Substrate
Km(l M ) kcat(s)1) kcat⁄ K m (s)1Æ M )1)
2.8 · 10 4
Trang 6MgCl2, 2 mm ATP, 0.5 mgÆmL)1BSA, 5 mm dithiothreitol,
1 lm [3H]dT and 0.5 ng Uu-TK or 0.2 lm [3H]dT and 1 ng
hTK1 in a total volume of 50 lL When the reaction was
finished, the filters were washed three times in 5 mm
ammo-nium formate and once in water Reaction products were
then eluted with 0.5 mL 0.1 m HCl⁄ 0.2 m KCl, and the
radioactivity was determined by liquid scintillation counting
(Beckman) The results were analysed by the SigmaPlot
Enzyme Kinetic Module version 1.1 (SPSS Science,
Chi-cago, IL, USA) The phosphoryl-transfer assay was
per-formed with 100 lm or 500 lm [c-32P]ATP in the same
buffer as above with variable concentrations of nucleosides
and 10 ng Uu-TK in a total volume of 25 lL The
phos-phorylated products were separated by TLC and quantified
by phosphorimaging analysis (Fujifilm Image Gauge,
ver-sion 3.3)
One unit of kinase activity was defined as the formation
of 1 nmol deoxyribonucleoside 5¢-monophosphate per mg
protein per min
Crystallization, data collection and refinement
The His-tag of Uu-TK was cleaved off overnight with
10 UÆmg)1 thrombin purchased from Amersham
Bio-sciences The protein was crystallized by the hanging drop
vapour diffusion method at 14C The protein solution
consisted of 10 mgÆmL)1 Uu-TK, 5 mm Thd and 3 mm
AMP-PCP, whereas the reservoir solution consisted of
15% poly(ethylene glycol) 3350 and 0.3 m ammonium
for-mate A small petri dish (diameter 5.5 cm) was filled with
0.5 mL of the crystallization solution 2 lL protein
solu-tion together with 2 lL crystallization solution was
applied to the lid of the Petri dish Two different crystal
forms appeared after 2–5 days Before flash-freezing in
liquid nitrogen, the crystals were swept through
cryo-pro-tecting solution consisting of 15% poly(ethylene glycol)
3350, 0.3 m ammonium formate and 20% glycerol
Data were collected at ID14-3 ESRF, processed with
mosflm [23], and scaled with scala from the CCP4
pro-gram suit [24] The statistics from data reduction are
pre-sented in Table 4 The structure was solved with molrep
[25] As search model, the tetramer of Uu-TK from the
dTTP complex, PDB code 1XMR, was used Residues 50–
67 were omitted from each chain of the search model
The omitted region or parts of the omitted region,
depending on the chain, could be traced with ARPwARP
[26] After simulated annealing performed in CNS,
subse-quent refinement was performed in REFMAC5 [27]
Dur-ing the whole refinement, NCS restraints were applied to
residues 12–49 and 69–213 for each protein chain By the
end of the refinement, the restraints were loosened to
medium for main-chain atoms and loose for side-chain
atoms A TLS model consisting of 15 TLS groups was
applied during the final steps of the refinement Each
monomer was divided into four domains [the a⁄ b domain,
the flexible loop (missing in subunit B), the lasso domain, and the C-terminus), together creating 15 TLS groups Table 4 shows the statistics from data refinement All model building was carried out in O [28] Each monomer contains 223 residues, although the N-terminus and C-ter-minus are disordered, and between 6–11 residues depend-ing on the chain are omitted from the model In addition, there is a disordered region between residues 51 and 66 Only in chain A could the whole region be traced; in chains C and D parts of the region are modelled, and in chain B the entire region is missing As observed previ-ously [14–16], this part of the structure takes different conformations The structure has been deposited with PDB code 2B8T
Acknowledgements
This work was supported by grants from the Swedish Research Council for the Environment, Agricultural Sciences and Spatial Planning (to L.Y and S.E.), the Swedish Research Council (to H.E and S.E.), and the Swedish Cancer Foundation (to H.E.)
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