Tài liệu Báo cáo khoa học: Functional studies of active-site mutants from Drosophila melanogaster deoxyribonucleoside kinase Investigations of the putative catalytic glutamate–arginine pair and of residues responsible for substrate specificity docx

melanogaster deoxyribonucleoside kinaseInvestigations of the putative catalytic glutamate–arginine pair and of residues responsible for substrate specificity Louise Egeblad-Welin1,2,*, Y

melanogaster deoxyribonucleoside kinase

Investigations of the putative catalytic glutamate–arginine pair and

of residues responsible for substrate specificity

Louise Egeblad-Welin1,2,*, Yonathan Sonntag1,*, Hans Eklund3 and Birgitte Munch-Petersen1

1 Department of Science, Systems and Models, Roskilde University, Denmark

2 Department of Molecular Biosciences, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden

3 Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden

Drosophila melanogaster deoxyribonucleoside kinase

(Dm-dNK) phosphorylates the four natural

deoxyribo-nucleosides, thymidine, deoxycytidine, deoxyadenosine

and deoxyguanosine, which is a crucial step in the

bio-synthesis of DNA precursors via the salvage pathway

In addition, Dm-dNK phosphorylates a number of important nucleoside analogue pro-drugs [1,2], making

it a potential candidate for use in suicide gene therapy

Keywords

catalytic mechanism; deoxyribonucleoside

kinase; dTTP; enzyme kinetics; nucleoside

analogues

Correspondence

B Munch-Petersen, Department of Science,

Systems and Models, Roskilde University,

Box 260, DK 4000 Roskilde, Denmark

Fax: +45 46743011

Tel: +45 46742418

E-mail: bmp@ruc.dk

L Egeblad-Welin, Department of Molecular

Biosciences, Swedish University of

Agricultural Sciences, Box 575, Biomedical

Center, S-751 25 Uppsala, Sweden

Fax: +46 18536971

Tel +46 184714192

E-mail: Louise.Egeblad@mbv.slu.se

*These authors contributed equally to this

work

(Received 2 November 2006, revised 4

January 2007, accepted 16 January 2007)

doi:10.1111/j.1742-4658.2007.05701.x

The catalytic reaction mechanism and binding of substrates was investi-gated for the multisubstrate Drosophila melanogaster deoxyribonucleoside kinase Mutation of E52 to D, Q and H plus mutations of R105 to K and

H were performed to investigate the proposed catalytic reaction mech-anism, in which E52 acts as an initiating base and R105 is thought to sta-bilize the transition state of the reaction Mutant enzymes (E52D, E52H and R105H) showed a markedly decreased kcat, while the catalytic activity

of E52Q and R105K was abolished The E52D mutant was crystallized with its feedback inhibitor dTTP The backbone conformation remained unchanged, and coordination between D52 and the dTTP–Mg complex was observed The observed decrease in kcatfor E52D was most likely due

to an increased distance between the catalytic carboxyl group and 5¢-OH of deoxythymidine (dThd) or deoxycytidine (dCyd) Mutation of Q81 to N and Y70 to W was carried out to investigate substrate binding The muta-tions primarily affected the Km values, whereas the kcatvalues were of the same magnitude as for the wild-type The Y70W mutation made the enzyme lose activity towards purines and negative cooperativity towards dThd and dCyd was observed The Q81N mutation showed a 200- and 100-fold increase in Km, whereas kcat was decreased five- and twofold for dThd and dCyd, respectively, supporting a role in substrate binding These observations give insight into the mechanisms of substrate binding and catalysis, which is important for developing novel suicide genes and drugs for use in gene therapy

Abbreviations

ACV, 9-(2-hydroxyethoxymethyl)-guanine; AraA, 9-(b- D -arabinofuranosyl)-adenine; AraC, 1-(b- D -arabinofuranosyl)-cytosine; AraT,

1-(b- D -arabinofuranosyl)-thymine; BVDU, (E)-bromvinyl-2¢-deoxyuridine; CdA, 2-chloro-2¢-deoxyadenosine; dAdo, deoxyadenosine; dCK, cytosolic deoxycytidine kinase; dCyd, deoxycytidine; dGK, deoxyguanosine kinase; dGuo, deoxyguanosine; Dm-dNK, Drosophila

melanogaster deoxyribonucleoside kinase; dThd, deoxythymidine; F-AraA, 2-flouro-9-(b- D -arabinofuranosyl)-adenine; FdUrd, 5-flouro-2¢-deoxyuridine; HSV1-TK, Herpes simplex virus Type 1 thymidine kinase; TK, thymidine kinase.

Sequence alignments and structural studies suggest

that Dm-dNK belongs to the family comprising

deoxy-guanosine kinase (dGK), deoxycytidine kinase (dCK),

thymidine kinase 2 (TK2) and herpes simplex virus

type 1 thymidine kinase (HSV1-TK) (Fig 1) [3] The

structures of Dm-dNK, human dGK and human dCK

were determined a few years ago [4,5]; although the structure of HSV1-TK has been known for several years, the first structures being solved in 1995 [6,7] The structure of human thymidine kinase 1 was solved recently, and was shown to belong to a structural fam-ily of its own [8,9]

Fig 1 Structural alignment of Dm-dNK, TK2, dGK, dCK and HSV1-TK Mutated amino acids are marked by their numbers in the Dm-dNK sequence Alignment of Dm-dNK, TK2, dGK and dCK was carried out with CLUSTALW (http://www.ebi.ac.uk/clustalw/) Alignment of HSV1-TK was carried out by structural comparison with the Dm-dNK structure in O [26].

Human deoxyribonucleoside kinases are targets for

the chemotherapeutic treatment of cancer and viral

diseases because they catalyse the addition of the first

phosphate group to the nucleoside analogue This

primes the nucleoside for further phosphorylation to

the corresponding triphosphate nucleoside, converting

the pro-drug to the active cytotoxic drug The

nucleo-side analogue can be incorporated into the DNA chain

and cause chain termination, induce apoptosis or

inhi-bit DNA polymerase [10,11] Because of the high

cata-lytic rate and the broad substrate specificity, it has

been suggested that Dm-dNK may be a putative

sui-cide gene in gene therapy In vivo experiments with

cancer cell lines showed increased sensitivity towards

nucleoside analogues [12] and a bystander effect was

observed [13,14]

The 3D structures of Dm-dNK, human dCK, human

dGK and HSV1-TK show a similar binding mode for

the substrates in the active site Three key residues in

Dm-dNK, identified and proposed as being responsible

for substrate specificity [4], were mutagenized and the

mutant enzymes characterized for their ability to

phos-phorylate native deoxyribonucleosides and nucleoside

analogues [15] These mutations of residues 84, 88 and

110 (Fig 2) converted dNK substrate specificity from

predominantly pyrimidine into purine

It has been suggested that the reaction mechanism

proposed for HSV1-TK [16] also applies to other

deoxy-ribonucleoside kinases [3] It is believed that E52 acts

as a base in the deprotonation of 5¢-OH, while the

transition state is stabilized by the positively charged R105 (Fig 2) The pKa of E52 is probably influenced

by the proximity of R105 which is high enough to act

as a base in the initial catalysis step In a structural study of Dm-dNK in which the enzyme was cocrystal-lized with both deoxythymidine (dThd) and dTTP sep-arately, E52 formed a hydrogen bond with the 5¢-OH group of dThd, whereas it was moved 6.5 A˚ in the dTTP complex and coordinated the Mg ion R105 is also affected; when dThd is bound R105 forms a hydrogen bond with E52, thus stabilizing the position and charge of E52, and when dTTP is bound it forms

a hydrogen bond with the a-phosphate of dTTP instead [17] Knowledge regarding the enzymatic reac-tion mechanisms is central to the design of mutant enzymes or nucleoside analogues for use in suicide gene therapy

We investigated the catalytic mechanism by muta-ting E52 to D, Q and H Provided that the pro-posed reaction mechanism [3] holds true, a profound effect on the catalytic rate should be evident from these mutations Binding of the substrates to the enzyme should not be altered to the same extent, as reflected by the lower impact on Km values Q is similar in size to E but cannot function as a base, whereas D and H should be able to act as a base but the differences in their pKa values and size may affect their efficiency Likewise, mutations of R105K and R105H to other positively charged residues are expected to influence catalytic rate rather than sub-strate binding

Two further active site residues responsible for substrate binding were investigated, these being Y70 and Q81 (Fig 2) These two amino acid residues are conserved among Dm-dNK, TK2, dGK, dCK and HSV1-TK (Fig 1) Y70 which anchors the 3¢-OH of the deoxyribose moiety of the nucleoside, together with E172 (Fig 2), was mutated to W This mutation was performed to see whether the larger side chain would affect substrate specificity Q81, which forms two hydro-gen bonds with the base, was mutated to N in order to see how the increased distance between the base and substrate-binding amino acid affected the binding of dThd and deoxycytidine (dCyd)

Results

We performed site-directed mutagenesis of four active site residues of Dm-dNK Residue E52 was mutated to

D, H and Q, residue Y70 to W, residue Q81 to N and residue R105 to K and H The kinetic properties of the active site mutants are summarized in Table 1 All mutants were characterized with dThd and dCyd,

Fig 2 Binding of the substrate dThd at the active site of Dm-dNK

[17] Hydrogen-bonding residues are shown E52, Y70, Q81 and

R105 were mutated in this study Residues V84, M88 and A110,

mutated in a previous study [15], are also included.

Y70W was further characterized with three nucleoside

analogues: 1-(b-d-arabinofuranosyl)-cytosine (AraC),

1-(b-d-arabinofuranosyl)-thymine (AraT) and

(E)-brom-vinyl-2¢-deoxyuridine (BVDU)

Mutants of catalytic residues: E52 and R105

It is evident from the kinetic results that mutation of

E52 changes only the catalytic rate For the E52D

mutant the Km value was approximately the same as

for the wild-type with dThd, whereas kcatwas 20 000

times lower This was also the scenario for the E52H mutant exhibiting kcat  1100 times lower than the wild-type The E52Q mutant did not show any meas-urable activity

Mutation of R105 also showed a decreased catalytic rate When mutated to K, the activity was lost com-pletely with both dThd and dCyd as substrates The R105H mutant showed a slightly increased Km value, sevenfold higher with dThd, and kcatwas decreased by

 2000-fold For dCyd as a substrate Kmwas 50-fold higher and kcatwas 275-fold lower

Table 1 Kinetics of dThd and dCyd phosphorylation for active-site mutants of Dm-dNK AraC, AraT and BVDU were tested only with the Y70W mutant k cat values were determined using a calculated mass of 26 785 kDa It is assumed that there is one active site per monomer Where cooperativity is observed, the Hill coefficient (n) is given When kcat⁄ K m is compared with the wild-type, dThd and dCyd is set to 100% Kinetic parameters were determined from three independent experiments, except where indicated by * or # , which were based on one or two experiments, respectively The results are given as mean ± SD ND, not detected.

Enzyme Substrate K m or K 0.5 (l M ) (n) V max (lmolÆmin)1Æmg)1) k cat (s)1) k cat ⁄ K m (s)1Æ M )1)

(100%)

(100%)

AraT 62 ± 7.4 10.4 ± 0.7 0.3 7 · 10 4

BVDU 2.2 ± 0.1 13.2 ± 1.7 5.9 2.6 · 10 6

E52D dThd 3.8 ± 0.6 0.00162 ± 0.00002 7.2 · 10)4 1.9 · 10 2

(< 1%) dCyd 3.7 ± 0.4 0.00160 ± 0.00018 7.1 · 10)4 1.9 · 10 2

(< 1%) E52H dThd* 3.7 0.02612 1.2 · 10)2 3.2 · 10 3

(< 1%) dCyd 5.8 ± 2.0 0.00259 ± 0.0006 1.2 · 10)3 2.0 · 10 2

(< 1%)

Y70W dThd# 251 ± 86

(n ¼ 0.6 ± 0.07)

5.2 ± 0.9 2.3 9.2 · 10 3

(< 1%)

dCyd 246 ± 34

(n ¼ 0.76 ± 0.002)

15.2 ± 0.3 6.8 2.8 · 10 4

(< 1%) AraC# 1441 ± 463 4.6 ± 0.6 2.1 1.4 · 10 3

(< 1%) AraT# 357 ± 43 2.3 ± 0.1 1.0 2.9 · 10 3

(< 1%) BVDU# 4.9 ± 0.4 0.82 ± 0.07 0.4 7.5 · 10 4

(< 1%) Q81N dThd 231 ± 24 12.1 ± 0.5 5.4 2.3 · 10 4

(< 1%) dCyd 205 ± 15 14.7 ± 2.0 6.6 3.2 · 10 4

(< 1%) R105H dThd 8.9 ± 1.5 0.015 ± 0.0008 6.7 · 10)3 7.5 · 10 2

(< 1%) dCyd 113 ± 24 0.124 ± 0.027 5.5 · 10)2 4.9 · 10 2

(< 1%)

a [2] b [15].

Mutants of substrate-binding residues: Y70 and

Q81

Y70W showed an increase in Kmvalues for both dThd

and dCyd, of approximately 200- and 100-fold,

respectively The kcat values decreased by

approxi-mately fivefold with dThd and twofold with dCyd An

interesting feature for this mutant is that it gained

neg-ative cooperativity with dThd and dCyd The Y70W

mutant was further characterized using the nucleoside

analogues, AraC, AraT and BVDU Kmfor AraC was

increased 59-fold, but this was less pronounced than

the increase of 106-fold for dCyd Vmax was fairly

unchanged compared with the wild-type The Kmvalue

for BVDU was increased approximately twofold

com-pared that for the wild-type, which indicated a minor

change in the binding of BVDU By contrast, kcatwas

decreased approximately 15-fold With AraT, Km

increased approximately sixfold and kcat decreased

approximately fivefold compared with the wild-type

Thus, for Y70W, the changes in Km with these

ana-logues were less pronounced than for dThd and dCyd

Overall, changing Y70 to W had a greater impact on

catalytic efficiency (kcat⁄ Km) with the natural substrates

than with the analogues Q81N had an increased Km

value for dThd and dCyd, of approximately 200- and

100-fold, respectively The kcat values were only

decreased approximately twofold for both substrates

Phosphorylation of nucleosides and nucleoside

analogues

Wild-type Dm-dNK and all mutants, with the exception

of E52H, were tested in a phosphotransferase assay

with natural nucleosides [dThd, dCyd, deoxyadenosine

(dAdo) and deoxyguanosine (dGuo)] and some

nucleo-side analogues [AraC, 2-chloro-2¢-deoxyadenosine (CdA),

2-flouro-9-(b-d-arabinofuranosyl)-adenine (F-AraA) and

5-flouro-2¢-deoxyuridine (FdUrd)] The mutant Y70W

was also investigated with some additional compounds

[dUrd, AraT, 9-(b-d-arabinofuranosyl)-adenine (AraA),

9-(2-hydroxyethoxymethyl)-guanine (ACV) and BVDU]

The catalytic mutants E52D, E52Q, R105H and R105K

did not show any detectable activity with either

nucleo-sides or nucleoside analogues (data not shown) in this

assay Only the wild-type, Y70W and Q81N were active

(see Table 2)

The most striking result for Y70W was that it

became an almost entirely pyrimidine-specific kinase,

because phosphorylation of dAdo and dGuo was

almost abolished, compared with the wild-type The

pyrimidine nucleoside analogues AraT, AraC, FdUrd

and BVDU were also phosphorylated by Y70W The

purine analogue CdA was phosphorylated but less effi-ciently compared with the wild-type, in accordance with the lowered activity with purines for Y70W Mutant Q81N was slightly less efficient towards the nucleosides compared with the wild-type In particular, phosphorylation of dGuo was reduced markedly

Structure of Dm-dNK-E52D in complex with dTTP The structure of one of the mutants, E52D, was solved using X-ray crystallography at a resolution of 2.5 A˚ in complex with the feedback inhibitor dTTP The R-fac-tor and Rfree were 23.2 and 24.2%, respectively (Table 3) Most of the protein could be found in the electron-density map, with the exception of resi-dues 1–11 and 210–230 The loop connecting a9 and b5 (residues 195–200) was flexible and poor density was observed The electron density for the mutant resi-due and the ligand was well defined Structural super-positioning of Dm-dNK-E52D–dTTP to the wild-type Dm-dNK–dTTP (PDB ID: 1OE0) was performed and showed an rmsd of 0.249 A˚2 over 358 Ca (for the dimer) This indicates that the E52D mutation does not change the overall structure of the enzyme, because the folds are almost identical D52 in this structure has a similar position to E52 in Dm-dNK– dTTP, i.e removed from the active site and binding a

Table 2 Nucleoside and nucleoside analogue phosphorylation by recombinant Dm-dNK mutant enzymes, using the phosphotransf-erase assay Relative levels of phosphorylation expressed in rela-tion to percentage dThd phosphorylarela-tion of the wild-type Relative phosphorylation expressed as a relation of the percentage of dThd phosphorylation of the corresponding mutant is given in paren-theses The substrate concentration is 100 l M Experiments were repeated twice with the exception of Q81N, which was assayed once Results are given as mean ± SD ND, not detected NI, not investigated.

Substrate ⁄

dThd (%) 100 78.4 ± 0.3 (100) 79.7 (100) dCyd (%) 87.7 ± 4.7 84.5 ± 3.1 (108) 87.9 (110) dAdo (%) 65.3 ± 2.3 2.5 ± 0.6 (3.2) 33.1 (41.5) dGuo (%) 38.4 ± 4.6 < 1 (< 1) 2.1 (2.6) dUrd (%) NI 56 ± 9 (71.4) NI AraT (%) NI 22 ± 4 (28.1) NI AraC (%) 52 ± 2 7 ± 2 (8.9) 1 (1.3) AraA (%) NI < 1 (< 1) NI F-AraA (%) 19 ± 6 ND 4 (5.0) CdA (%) 120 ± 11 11.5 ± 0.5 (14.7) 96 (120)

FdUrd (%) 48 ± 0 37 ± 8 (47.2) 23 (28.9) BVDU (%)a 54 19.5 ± 8.5 (24.9)

a

[15].

Mg ion that coordinates the phosphates of dTTP

(Fig 3)

Discussion

Mutation of residues in the active site was intended to:

(a) validate the proposed reaction mechanism [3] by

mutating the putative catalytic base (E52) and arginine (R105), thought to stabilize the transition state and holding E52 in position during catalysis; and (b) investigate the amino acid residues involved in sub-strate binding (Y70 and Q81) The steady-state kinetics

of Dm-dNK is compulsory ordered with formation of

a ternary complex [1,2] Pre-steady-state measurements indicate that either the catalytic step or a preceding step is rate determining for the overall forward reac-tion (R Browne, G Andersen, G Le, B Munch-Petersen and C Grubmeyer, unpublished results) Therefore, and because ATP is saturating in our experiments, when evaluating the impact of the muta-tions from the kinetic data, a change in the Km value can be interpreted as an effect on substrate binding, and a change in kcat would reflect an effect on the catalytic step

E52 mutations E52D

The point mutation E52D was investigated using both kinetic and structural studies Kinetic results with an unchanged Kmvalue indicated that the affinity for sub-strates was unchanged, whereas catalytic activity was altered because the kcat value had decreased dramatic-ally The structural study showed that the backbone conformation of the enzyme was unchanged Because the chemical properties of Glu and Asp are very sim-ilar, both having a carboxylic acid functional group, the dramatically decreased catalytic rate is most likely due to the increased distance between the catalytic base and the 5¢-OH of either dThd or dCyd These results favour the reaction-mechanistic hypothesis’ emphasis on arginine and glutamate acting as a pair in the phosphorylation

E52H Mutation of E52 to H resulted in a greatly reduced

kcat value, although the Km value was relatively unchanged With its imidazole ring, histidine can act

as both a proton donor and an acceptor in enzymatic reactions, and it should therefore theoretically be able

to replace glutamate as a base, to some extent How-ever, this is not the case One reason may be an altered local conformation, because the normal hydrogen bond network will be affected Modelling mutation of E52H into the structure of wild-type Dm-dNK with dCyd bound (PDB ID: 1J90) reveals an increase in the distance between the 5¢-OH group and histidine of

 1 A˚ relative to glutamate This alone could explain the reduced catalytic rate

Table 3 Data collection and refinement statistics for the dNK-E52D

in complex with dTTP

Dm-dNK E52D

Cell dimensions (A ˚ ) a ¼ 33.5

b ¼ 119.5

c ¼ 68.9

b ¼ 92.42

Content of asymmetric unit One dimer

Resolution range (A ˚ ) 32.3–2.5

Completeness (%) 100.0 (100.0)

Number of unique reflections 18,797

Wavelength (A ˚ ) 1.087

Temperature (K) 100

rmsd

Bond length (A ˚ ) 0.007

Bond angles () 0.912

Mean B-value (A˚2 ) 37.1

Fig 3 Structural alignment of wild-type Dm-dNK (blue) (PDB ID:

1OE0) and Dm-dNK-E52D (red) illustrating the binding of the

feed-back inhibitor dTTP and the position of Mg 2+ in the active site.

To further support to the hypothesis of the role of E52

as a proton abstractor it was also mutated to its amide –

glutamine This mutant did not show any detectable

activity with either dThd or dCyd The two amino acids

take up roughly the same volume, the position of the

side chain can be expected to occupy roughly the same

position and glutamine can participate in hydrogen

bonding The result that E52Q did not show any activity

must therefore be a consequence of the reactivity of the

functional group and further support the hypothesis

that E52 acts as an initiating base in the reaction

R105 mutations

R105H

R105 is thought to stabilize the transition state and

hold E52 in the correct position to initiate the catalytic

reaction The transition state of this type of kinases is

considered to be close to trigonal bipyramidal

geo-metry of the phosphate to be transferred Arg105 as

well as a Mg ion and arginines of the Lid-region

sta-bilize the negative phosphates of this state The most

striking effect of mutation of arginine to histidine is an

almost 2000-fold decrease in the kcat value for dThd,

and a 270-fold decrease for dCyd The Km value was

increased sevenfold for dThd and 49-fold for dCyd

The large decrease in kcat value indicates that Arg

plays an important role in catalysis Arginine adopts

two distinct conformations depending on whether a

substrate or an inhibitor is bound [17] When mutated

to histidine, the residue is no longer able to make

the same hydrogen bonds in the different states The

decrease in kcatand increase in Km may be due to the

bulky and rigid structure of histidine that can cause

steric hindrances for the substrate, wherefore correct

positioning and stabilization of the negative charge of

E52 will be less than optimal

R105K

Surprisingly, mutation of R105 to K completely

abol-ishes the catalytic efficiency A possible explanation for

this is that lysine is more flexible than Arg, and therefore

not able to position E52, and catalyse the reaction

Mutations of substrate-interacting residues:

Y70W and Q81N

Y70W

When Y70 was mutated to W the kinetic results

showed that the Km values with dThd and dCyd were

dramatically increased, whereas the kcat values were only slightly decreased Thus, the mutation primarily affects binding of the substrate A similar point muta-tion was made in HSV1-TK, namely Y101 to F In this study, the Km value for HSV1-TK-Y101F was increased 12.5-fold, whereas the kcatvalue was twofold lower [18] The structure of HSV1-TK-WT was deter-mined in complex with (North)-methanocarba-thymidine,

as was the structure of HSV1-TK-Y101F A structural superposition showed that there were no significant changes in the polypeptide chain, except that the hydrogen bond from Y101–3¢-OH was lost [18] Based

on our results and the information from the structures

of HSV1-TK we suggest that the network of hydrogen bonds is disrupted, and this gives rise to an increase in

Km Also, the polarity is changed, and the increase in the size of the side chain may create steric hindrance for the substrate, making the base moiety of the sub-strates bind in a nonoptimal conformation

Another interesting feature concerning the Y70W mutant is that it became almost entirely pyrimidine spe-cific, which is also true for the nucleoside analogues This must be a consequence of the tryptophan creating steric hindrance for the purines The intention behind the mutation of Y70 to W was to create an enzyme with increased affinity towards ACV, because tryptophan was thought to make a better fit for the acyclic ribose moiety in the active site compared with the bulkier ribose ring of naturally occurring nucleosides However,

no activity with ACV was detected for Y70W The pres-ence of the bulky dGuo base in ACV may be the reason When the kinetic constants were determined for Y70W with the analogues AraT, AraC and BVDU, it was surprising that BVDU had a very low Km value (4.9 lm,  50-fold lower than the Km value for dThd and dCyd) The crystal structure of Dm-dNK with var-ious substrates shows that there is a deep hydrophobic pocket at the 5-position of the base The bromovinyl group may interact with the amino acids lining this space Binding of BVDU to Dm-dNK-Y70W compen-sates for the loss of one hydrogen bond to 3¢-OH by a tighter fitting of the bromovinyl group, thus restoring the tight binding lost due to the mutation At the same time, it is possible that the positioning of 5¢-OH is chan-ged, and this may be the reason for the low kcatvalue

Q81N The kinetic data for Q81N show a dramatic increase

in the Km values, whereas kcat is decreased slightly These results suggest that the tight anchoring of the base (dThd or dCyd) is lost, thereby resulting in poorer binding and higher Kmvalues

The relative phosphorylation of dAdo and dGuo

also showed a significant decrease compared with the

wild-type, most likely because of poorer binding of the

purine substrates

In a previous study of HSV1-TK, Q125 (equivalent to

Q81 in Dm-dNK) was point mutated to N The

HSV1-TK-Q125N were solved in complex with dThd, and the

main difference between the two structures was that the

tight binding of dThd (wild-type) was replaced by a

single water-mediated hydrogen bond (Q125N) Their

kinetic data supported this, because the Km value for

dThd with HSV1-TK-Q125N was increased 50-fold and

the catalytic rate was not affected, seen in comparison

with the wild-type [19] The results obtained in this study

together with the information from HSV1-TK suggest

that the increased distance between base and amino acid

is the main reason for the increase in Km values The

point mutation probably does not alter the backbone

conformation of the Ca-atoms

In conclusion, the kinetic and structural data

presen-ted here, emphasizing the role of R105 and E52 in the

catalytic mechanism, have gained further support

Together with findings of substrate-binding

interac-tions via the Q81N and Y70W mutainterac-tions, additional

knowledge about the structure–function relationship of

the ultra fast Dm-dNK has been obtained

Experimental procedures

Materials

Glutathione–Sepharose, pGEX-2T vector, Escherichia coli

strain BL21(DE3)pLysS, thrombin, [methyl-3H]thymidine

(25 CiÆmmol)1), [5–3H]-deoxycytidine (24 CiÆmmol)1) and

[32P]ATP[cP] (3000 CiÆmmol)1) were purchased from

Amer-sham Biosciences (Uppsala, Sweden) BVDU (14.3 CiÆmmol)1),

1-b-d-arabinofuranosyl thymine (2.89 CiÆmmol)1) and

1-b-d-arabinofuranosyl cytosine (23.30 CiÆmmol)1) were from

Moravek Biochemicals Inc (Brea, CA) Radiolabelled

nucle-osides were diluted with the nonradioactive compounds to

the appropriate concentrations When present in the

radiola-belled deoxynucleosides, ethanol was evaporated before use

Non-radioactive nucleosides were from Sigma Materials for

cloning, PCR, DNA sequencing, assay and crystallization

were standard commercially available products

Site-directed mutagenesis and expression

plasmid

Expression plasmid pGEX-2T-Dm-dNK has been described

previously [2] All mutants were constructed using

site-directed mutagenesis on the plasmid pGEX-2T-Dm-dNK

with truncation for 20 terminal amino acids The primers used to create the point mutations, where the changed nucleotides are in boldface and underlined, are as follows: E52D-fwd: 5¢-GCCTGCTGACCGACCCCGTCGAGAAG TGGCGC-3¢ E52D-rev: 5¢-GCGCCACTTCTCGACGGG GTCGGTCAGCAGGC-3¢ E52H-fwd: 5¢-GCCTGCTGAC CCACCCCGTCGAGAAGTGGCGC-3¢ E52H-rev: 5¢-GC GCCACTTCTCGACGGGGTGGGTCAGCAGGC-3¢

CGC-3¢ E52Q-rev: 5¢-GCGCCACTTCTCGACGGGCTG GGTCAGCAGGC-3¢ Y70W-fwd: 5¢-CTGCTGGAGCT GATGTGGAAAGATCCCAAGAAG-3¢ Y70W-rev: 5¢-CTT CTTGGGATCTTTCCACATCAGCTCCAGCAG-3¢ Q81N-fwd: 5¢-TGGGCCATGCCCTTTAACAGTTATGTCACG CTG-3¢ Q81N-rev: 5¢-CAGCGTGACATAACTGTTAAA GGGCATGGCCCA-3¢ R105H-fwd: 5¢-GCTAAAAATAA

R105H-rev: 5¢-GCGAGCGCTAAAAATGGAGTGCTCCATTAT TTTTAGC-3¢ R105K-fwd: 5¢-GCTAAAAATAATGGAG AAATCCATTTTTAGCGCTCGC-3¢ R105K-rev: 5¢-GCG AGCGCTAAAAATGGATTTCTCCATTATTTTTAGC-3¢

Sequence verification

Plasmids of the seven mutants were transformed into XL1-Blue Supercompetent Cells Plasmids were isolated and the insert sequenced using the dye terminator method (ABI PRISM 310), in order to verify that the point mutations were introduced, and that no other mutations or frame-shifts had occurred

Expression and purification

The seven pGEX-2T Dm-dNKDC20 mutants were trans-formed into E coli BL21-competent cells Recombinant proteins were expressed and purified and thrombin was cleaved as described previously [2]

All proteins were stored at)80 C, and a cryoprotectant solution was added to a final concentration of: 10% (v⁄ v) glycerol, 0.1% (v⁄ v) Triton X-100, 5 mm MgCl2 and 5 mm dithiothreitol, with the exception of Dm-dNK E52D CD20;

it was stored in 30% glycerol

The purity of the proteins was determined by SDS⁄ PAGE [20] and the protein concentrations were deter-mined using Bradford reagent [21]

Enzyme assays

Deoxynucleoside kinase activities were determined by initial velocity measurements based on four time samples (0, 4,

8 and 12 min) using the DE-81 filter paper assay with trit-ium-labelled substrates as described previously [2]

The standard assay conditions were: 50 mm Tris⁄ HCl

pH 7.5, 2.5 mm MgCl2, 10 mm dithiotreitol, 0.5 mm

CHAPS, 0.5 mgÆmL)1 bovine serum albumin and 2.5 mm

ATP

The relative phosphorylation of nucleosides and

nucleo-side analogues was determined using the phosphoryl transfer

assay This was performed using [32P]ATP[cP] The

nucleo-sides⁄ analogues were added to a final concentration of

100 lm in a reaction mixture of 25 lL The standard reaction

buffer contained 50 mm Tris–HCL pH 7.5, 2.5 mm MgCl2,

10 mm dithiothreitol, 0.5 mm CHAPS, 0.5 mm bovine serum

albumin, 100 lm nonradioactive labelled ATP, radioactively

labelled ATP, 50 ng enzyme per reaction After incubation of

the reaction mixtures for 20 min at 37C, 1 lL was spotted

on a TLC sheet The nucleotides were separated in a buffer

containing NH4OH, isobutyric acid and destilled H2O in a

ratio of 1:66:33 (v⁄ v ⁄ v) Sheets were autoradiographed using

phosphorimaging plates

The kinetic data were evaluated using nonlinear

regres-sion analysis and the Michaelis–Menten equation v¼

VmaxÆ[S]⁄ (Km+ [S]) or the Hill equation v¼ VmaxÆ[S]n⁄

(Kn

0:5+ [S]n) as described previously [22] All kinetic data

were analysed using sigma plot

Crystallization

Crystals of a C-terminally truncated (D20) recombinant

Dm-dNK mutant E52D were grown using the vapour

diffu-sion method by hanging drop geometry The crystallization

solution was: 0.12 m NaAc pH 7.0, 0.1 m Mes pH 6.5 and

18% (w⁄ v) monomethyl polyethylene glycol 2000 The

enzyme solution consisted of 5 mgÆmL)1mutant enzyme in

a 1· NaCl ⁄ Pibuffer with 5 mm dTTP, 5 mm Mg2+, 5 mm

dithiothreitol and 10% glycerol The crystallization solution

was diluted 1:2 with water before 2 lL was mixed with

2 lL enzyme solution on a cover slip The well solution

was covered with 250 lL Al’s Oil It was left to equilibrate

against the crystallization solution at 15C After

approxi-mately 3 days, small crystals appeared and after 5 days

larger singular crystals were obtained

Data collection

The E52D–dTTP crystals were flash-frozen in liquid

nitro-gen The cryoprotectant had the same composition as the

crystallization solution plus an added 20% (v⁄ v) glycerol

The data set was collected at MAXLab in Lund, Sweden,

on beam line I711 at a temperature of 100 K The data

were indexed scaled and merged using mosflm [23] and

scala [24] The crystal belonged to the monoclinic space

group P21and had a solvent content of 48%

Structure determination and refinement

The structure was solved by molecular replacement using

molrep [25] with the wild-type structure Dm-dNK-dTTP

(PDB ID: 1OE0) as the search model The mutated residue was replaced using o v 9.0.7 (http://xray.bmc.uu.se/alwyn) [26], and rigid body refinement was performed using refmac5 [27] Constrained refinement with a twofold non-crystallographic symmetry was carried out using refmac5 Water molecules, Mg2+ and the ligand dTTP were added using the program o [26] Data collection and refinement statistics are shown in Table 3 The coordinates have been deposited with the PDB ID: 2jcs

Acknowlegdements

This work was supported by grants from the Swedish Research Council (to HE), the Swedish Cancer Foun-dation (to HE), the Danish Research Council (to BM-P) and the NOVO Nordisk foundation (to BM-P)

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