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

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 Louise Egeblad-Welin 1,2,* , Yonathan Sonntag 1,* , Hans Eklund 3 and Birgitte Munch-Petersen 1 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 k cat , 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 k cat for 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 K m values, whereas the k cat values 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 K m , whereas k cat 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. 1542 FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 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]. L. Egeblad-Welin et al. Active site mutation of deoxyribonucleoside kinase FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 1543 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 pK a 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 K m 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 pK a 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. Active site mutation of deoxyribonucleoside kinase L. Egeblad-Welin et al. 1544 FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 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 K m value was approximately the same as for the wild-type with dThd, whereas k cat was  20 000 times lower. This was also the scenario for the E52H mutant exhibiting k cat  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 K m value, sevenfold higher with dThd, and k cat was decreased by  2000-fold. For dCyd as a substrate K m was  50-fold higher and k cat was 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 k cat ⁄ 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 (lM) (n) V max (lmolÆmin )1 Æmg )1 ) k cat (s )1 ) k cat ⁄ K m (s )1 ÆM )1 ) Wild-type a dThd 1.2 29.5 14.2 1.2 · 10 7 (100%) dCyd 2.3 34.2 16.2 7.0 · 10 6 (100%) AraC b 24.3 5.6 2.9 1.2 · 10 5 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%) E52Q dThd ND ND – – dCyd ND ND – – 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%) R105K dThd ND ND – – dCyd ND ND – – a [2]. b [15]. L. Egeblad-Welin et al. Active site mutation of deoxyribonucleoside kinase FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 1545 Mutants of substrate-binding residues: Y70 and Q81 Y70W showed an increase in K m values for both dThd and dCyd, of approximately 200- and 100-fold, respectively. The k cat 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. K m for AraC was increased 59-fold, but this was less pronounced than the increase of 106-fold for dCyd. V max was fairly unchanged compared with the wild-type. The K m value 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, k cat was decreased approximately 15-fold. With AraT, K m increased approximately sixfold and k cat decreased approximately fivefold compared with the wild-type. Thus, for Y70W, the changes in K m with these ana- logues were less pronounced than for dThd and dCyd. Overall, changing Y70 to W had a greater impact on catalytic efficiency (k cat ⁄ K m ) with the natural substrates than with the analogues. Q81N had an increased K m value for dThd and dCyd, of approximately 200- and 100-fold, respectively. The k cat 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 R free 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 phosphorylation 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 ⁄ Enzyme WT Y70W Q81N 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) ACV (%) NI ND NI FdUrd (%) 48 ± 0 37 ± 8 (47.2) 23 (28.9) BVDU (%) a 54 19.5 ± 8.5 (24.9) a [15]. Active site mutation of deoxyribonucleoside kinase L. Egeblad-Welin et al. 1546 FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 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 K m value can be interpreted as an effect on substrate binding, and a change in k cat 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 K m value indicated that the affinity for sub- strates was unchanged, whereas catalytic activity was altered because the k cat 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 k cat value, although the K m 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  1A ˚ 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 Space group P2 1 Cell dimensions (A ˚ )a¼ 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) R sym (%) 9.1 (31.1) I ⁄ rI 6.9 (2.3) Redundancy 3.8 (3.8) Number of unique reflections 18,797 Beamline I711 Wavelength (A ˚ ) 1.087 Temperature (K) 100 R-factor (%) 23.2 R free (%) 24.2 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. L. Egeblad-Welin et al. Active site mutation of deoxyribonucleoside kinase FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 1547 E52Q 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 k cat value for dThd, and a 270-fold decrease for dCyd. The K m value was increased sevenfold for dThd and 49-fold for dCyd. The large decrease in k cat 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 k cat and increase in K m 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 K m values with dThd and dCyd were dramatically increased, whereas the k cat 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 K m value for HSV1-TK-Y101F was increased 12.5-fold, whereas the k cat value 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 K m . 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 K m value (4.9 lm,  50-fold lower than the K m 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 k cat value. Q81N The kinetic data for Q81N show a dramatic increase in the K m values, whereas k cat 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 K m values. Active site mutation of deoxyribonucleoside kinase L. Egeblad-Welin et al. 1548 FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 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 struc- tures of both wild-type HSV1-TK and 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 K m 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 K m 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 mutations, 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- 3 H]thymidine (25 CiÆmmol )1 ), [5– 3 H]-deoxycytidine (24 CiÆmmol )1 ) and [ 32 P]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¢-GCCTGCTGACCGA CCCCGTCGAGAAG TGGCGC-3¢. E52D-rev:5¢-GCGCCACTTCTCGACGGG GTCGGTCAGCAGGC-3¢. E52H-fwd:5¢-GCCTGCTGAC C CACCCCGTCGAGAAGTGGCGC-3¢. E52H-rev:5¢-GC GCCACTTCTCGACGGG GTGGGTCAGCAGGC-3¢. E52Q- fwd:5¢-GCCTGCTGACC CAGCCCGTCGAGAAGTGG CGC-3¢. E52Q-rev:5¢-GCGCCACTTCTCGACGGGCT G GGTCAGCAGGC-3¢. Y70W-fwd:5¢-CTGCTGGAGCT GATGT GGAAAGATCCCAAGAAG-3¢. Y70W-rev :5¢-CTT CTTGGGATCTTT CCACATCAGCTCCAGCAG-3¢. Q81N- fwd:5¢-TGGGCCATGCCCTTT AACAGTTATGTCACG CTG-3¢. Q81N-rev:5¢-CAGCGTGACATAACT GTTAAA GGGCATGGCCCA-3¢. R105H-fwd:5¢-GCTAAAAATAA TGGAGC ACTCCATTTTTAGCGCTCGC-3¢ . R105H- rev:5¢-GCGAGCGCTAAAAATGGAG TGCTCCATTAT TTTTAGC-3¢. R105K-fwd:5¢-GCTAAAAATAATGGAG AAATCCATTTTTAGCGCTCGC-3¢. R105K-rev:5¢-GCG AGCGCTAAAAATGGA TTTCTCCATTATTTTTAGC-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 MgCl 2 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 MgCl 2 ,10mm dithiotreitol, 0.5 mm L. Egeblad-Welin et al. Active site mutation of deoxyribonucleoside kinase FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 1549 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 [ 32 P]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 MgCl 2 , 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 37 °C, 1 lL was spotted on a TLC sheet. The nucleotides were separated in a buffer containing NH 4 OH, isobutyric acid and destilled H 2 Oina 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 v ¼ V max Æ[S] ⁄ (K m +[S]) or the Hill equation v ¼ V max Æ[S] n ⁄ (K n 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 )1 mutant enzyme in a1· NaCl ⁄ P i buffer with 5 mm dTTP, 5 mm Mg 2+ ,5mm 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 15 °C. 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 P2 1 and 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, Mg 2+ 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) References 1 Munch-Petersen B, Piskur J & Søndergaard L (1998) Four deoxynucleoside kinase activities from Drosophila melanogaster are contained within a single monomeric enzyme, a new multifunctional deoxynucleoside kinase. J Biol Chem 273, 3926–3931. 2 Munch-Petersen B, Knecht W, Lenz C, Søndergaard L & Piskur J (2000) Functional expression of a multisub- strate deoxynucleoside kinase from Drosophila melano- gaster and its C-terminal deletion mutants. J Biol Chem 275, 6673–6679. 3 Eriksson S, Munch-Petersen B, Johansson K & Eklund H (2002) Structure and function of cellular deoxyribo- nucleoside kinases. Cell Mol Life Sci 59, 1327–1346. 4 Johansson K, Ramaswamy S, Ljungcrantz C, Knecht W, Piskur J, Munch-Petersen B & Eklund H (2001) Structural basis for the substrate specificities of cellular deoxynucleoside kinases. Nat Struct Biol 8, 616–620. 5 Sabini E, Ort S, Monnerjahn C, Konrad M & Lavie A (2003) Structure of human dCK suggest strategies to improve anticancer and antiviral therapy. Nat Struct Biol 10, 513–519. 6 Wild K, Bohner T, Aubry A, Folkers G & Schulz GE (1995) The three-dimensional structure of thymidine kinase from herpes simplex virus type 1. FEBS Lett 168, 289–292. 7 Brown DG, Visse R, Sandhu G, Davies A, Rixkallah PJ, Melitz C, Summers WC & Sanderson MR (1995) Crystal structures of the thymidine kinase from herpes simplex virus type-1 in complex with deoxythymidine and ganciclovir. Nat Struct Biol 4, 2316–2320. 8 Welin M, Kosinska U, Mikkelsen NE, Carnrot C, Zhu C, Wang L, Eriksson S, Munch-Petersen B & Eklund H (2004) Structures of thymidine kinase 1 of human and mycoplasmic origin. Proc Natl Acad Sci USA 101, 17970–17975. Active site mutation of deoxyribonucleoside kinase L. Egeblad-Welin et al. 1550 FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 9 Birringer MS, Claus MT, Folkers G, Kloer DP, Schulz GE & Scapozza L (2005) Structure of a type II thymi- dine kinase with bound dTTP. FEBS Lett 579, 1376– 1382. 10 Robak T, Korycka A, Kasznicki M, Wrzesien-Kus A & Smolewski P (2005) Purine nucleoside analogues for the treatment of hematological malignancies: pharmacology and clinical applications. Curr Cancer Drug Targets 5, 421–444. 11 Munch-Petersen B & Piskur J (2006) Deoxynucleoside kinases and their potential role in deoxynucleoside cyto- toxicity. In Cancer Drug Discovery and Development: Deoxynucleoside Analogs in Cancer Therapy (Peters GJ, ed.), pp. 53–79. Humana Press, Totowa, NJ. 12 Solaroli N, Johansson M, Balzarini J & Karlsson A (2007) Enhanced toxicity of purine nucleoside analogs in cells expressing Drosophila melanogaster nucleoside kinase mutants. Gene Ther 14, 86–92. 13 Zheng X, Johansson M & Karlsson A (2000) Retro- viral transduction of cancer cell lines with the gene encoding Drosophila melanogaster multisubstrate deoxyribonucleoside kinase. J Biol Chem 275, 39125– 39129. 14 Zheng X, Johansson M & Karlsson A (2001) Nucleo- side analog cytotoxicity and bystander cell killing of cancer cells expressing Drosophila melanogaster deoxy- ribonucleoside kinase in the nucleus or cytosol. Biochem Biophys Res Commun 289, 229–233. 15 Knecht W, Sandrini MP, Johansson K, Eklund H, Munch-Petersen B & Pis ˇ kur J (2002) A few amino acid substitutions can convert deoxyribonucleoside kinase specificity from pyrimidines to purines. EMBO J 21, 1873–1880. 16 Wild K, Bohner T, Folkers G & Schulz GE (1997) The structure of thymidine kinase from herpes simplex type 1 in complex with substrates and a substrate analogue. Protein Sci 6, 2097–2106. 17 Mikkelsen NE, Johansson K, Karlsson A, Knecht W, Andersen G, Piskur J, Munch-Petersen B & Eklund H (2003) Structural basis for feedback inhibition of the deoxyribonucleoside salvage pathway: studies of the Drosophila deoxyribonucleoside kinase. Biochemistry 44, 5706–5712. 18 Prota A, Vogt J, Pilger B, Perozzo R, Wurth C, Mar- quez VE, Russ P, Schulz GE, Folkers G & Scapozza L (2000) Kinetic and crystal structure of the wild-type and engineered Y101F mutant of herpes simplex virus type 1 thymidine kinase interacting with (north)-methano- carba-thymidine. Biochemistry 39, 9597–9603. 19 Vogt J, Perozzo R, Pautsch A, Prota A, Schelling P, Pilger B, Folkers G, Scapozza L & Schulz GE (2000) Nucleoside binding site of herpes simplex type 1 thymi- dine kinase analyzed by X-ray crystallography. Proteins 41, 545–553. 20 Laemmli UK (1970) Cleavage of structural proteins dur- ing the assembly of the head bacteriphage T4. Nature 227, 680–685. 21 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein util- izing the principle of protein–dye binding. Anal Biochem 72, 248–254. 22 Knecht W, Bergjohann U, Gonski S, Kirschbaum B & Loffler M (1996) Functional expression of a fragment of human dihydroorotate dehydrogenase by means of the baculovirus expression vector system, and kinetic inves- tigation of the purified recombinant enzyme. Eur J Bio- chem 240, 292–301. 23 Leslie AGW (1992) Joint CCP4 + ESF-EAMCB News- letter on Protein Crystallography, no. 26. 24 Collaborative Computational Project Number 4 (1994) The CCP4 Suite: programs for protein crystallography. Acta Crystallogr D50, 760–763. 25 Vagin A & Teplyakov A (2000) An approach to multi- copy search in molecular replacement. Acta Crystallogr D Biol Crystallogr 56, 1622–1624. 26 Jones TA, Zou JY, Cowan SW & Kjeldgaard M (1991) Improved methods for building protein models in elec- tron density maps and the location of errors in these models. Acta Crystallogr 47, 110–119. 27 Murshudov GN, Vagin AA & Dodson EJ (1997) Refinement of macromolecular structures by maximum- likelihood method. Acta Crystallogr D53, 240–255. L. Egeblad-Welin et al. Active site mutation of deoxyribonucleoside kinase FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 1551 . Functional studies of active-site mutants from Drosophila melanogaster deoxyribonucleoside kinase Investigations of the putative catalytic glutamate–arginine. 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.