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A novel viral thymidine monophosphate kinase with diphosphate kinase activity
Eduardo Guevara-Hernandez 1, Aldo A. Arvizu-Flores 2,* , Enrique F. Velazquez- Contreras 2, Francisco J. Castillo-Yañez 2, Luis G. Brieba 3, Rogerio R. Sotelo-Mundo 1,*
1 Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD). Carretera a Ejido La Victoria Km 0.6, Apartado Postal 1735, Hermosillo, Sonora, 83304, México.
2 Universidad de Sonora, Departamento de Ciencias Químico Biológicas. Blvd. Rosales y Luis Encinas, Hermosillo, Sonora, 83000 México.
3 Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de Investigación y Estudios Avanzados-Unidad Irapuato Km 9.6 Libramiento Norte Carretera Irapuato-León; Apartado Postal 629; Irapuato, Guanajuato, 36500, México,
69 ABSTRACT
Nucleotide phosphorylation is a key step towards DNA replication and during viral infections the maintenance of the nucleotide triphosphates pool is required. Deoxi thymidine triphosphate (dTTP) is produced either by de novo or salvage pathways.
Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates deoxi thymidine monophosphate (dTMP) using adenosine triphosphate (ATP) as a phosphate donor. In the white spot syndrome virus (WSSV) there is an open reading frame that encodes in a single polypeptide a thymidine kinase and TMK. We overexpressed the TMK domain, which was also able to use dTDP as a substrate to produce dTTP. White spot syndrome virus TMK (TMKwssv) has presented a sequential ordered bi bi mechanism of reaction and the efficiency of production of dTDP by TMKwssv is one of the highest compared to other organisms.
The affinity constants of TMKwssv substrates studied by kinetics were compared to a nucleosidic analog. The affinity for d4TMP was similar to the natural substrate. These results suggest that it is possible to propose the use of these compounds as drugs to treat this kind of viral diseases in shrimp.
INTRODUCTION
Novel pathogens appear to have interesting strategies in terms of their non-structural genes. In crustaceans there is a double stranded circular DNA virus of about 300 kb (van Hulten et al., 2001) that encodes multiple DNA metabolisms genes such as a DNA polymerase (de-la-Re-Vega et al., 2011), ribonucleotide reductase (van Hulten et al.,
70 2000), thymidylate synthase (Arvizu-Flores et al., 2009) and a bifunctional open reading frame that encodes in a single polypeptide a thymidine kinase (TK) and a thymidine monophosphate kinase (TMK) (Tsai et al., 2000). In between of those two domains there is a stretch of lysines that make feasible to predict a proteolytic cleavage in vivo to lead to two monomers that can oligomerize to give functional proteins. Recombinant overexpression of the complete ORF leads to a protein that had only the TK enzymatic activity (Tzeng et al., 2002). Other viral TMKs studied are those from vaccinia poxvirus (Caillat et al., 2008), Chilo iridescent virus (Jakob et al., 2001) and one recently identified in Erwinia phage Ea35-70 (GenBank entry AHI60461.1). The objective of this paper, was to study the kinetic and thermodynamic properties of TMKwssv, in order to describe catalytic properties of the enzyme. Since this virus also encodes its own thymidylate synthase, it is possible to envision that it can provide its own dTTP for viral DNA replication.
MATERIALS AND METHODS
TMK expression and purification
A synthetic construct containing the TMK domain (residues 201 to 398) from the bifunctional TK-TMK from white spot syndrome virus (GenBank: NP_477917), was used to overexpress the functional TMKwssv. Details of the recombinant expression and purification have been reported elsewhere (Guevara-Hernandez et al., 2012).
Identification of phosphorylation products by thin layer chromatography (TLC)
71 TLC was used to determinate specific thymidine monophosphate kinase activity.
The reaction buffer contained 50 mM Tris HCl pH 7.5, 2 mM MgCl2, 0.2 mM dTMP, 0.5 mg/mL of BSA, 1 mM DTT, and 15 mM NaF. The reaction was started by addition of 1 μCi of [γ32-P]ATP and 40 ng of TMKwssv in a final reaction volume of 20 μL. The reaction was stopped at different times (2, 4, 8 and 16 minutes) by heating aliquots at 70º C for 2 minutes. 2 μL of each aliquot was spotted onto TLC sheet (PEI cellulose 20 x 20 cm Merck Millipore) and samples were eluted inside a TLC chamber with 0.5 M of ammonium formate pH 3.5 as eluent. A Storm 820 phosphorimager (Amersham Bioscience) and Quantity-One software were used to analyze images.
Identification of phosphorylation products by high-performance liquid chromatography (HPLC)
Specific activity of nucleotide diphosphate kinase of TMKwssv were determinate using an HPLC system series 1100 and a UV cell detector series 1100 (Agilent Technologies, Waldbronn, Germany). Detection of dTTP formation was follow as described (Ryder 1985). Reaction done in 25 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM ATP and 1 mM dTDP, and was started by adding 50 μg of TMKwssv (quantified by BCA method) in a final volume of 1 mL, at 25º C and 100 rpm of orbital agitation. Reaction samples were deactivated by adding 0.6 M of perchloric at 1, 2 and 3 hours, respectively. Each sample was centrifuged at 16,000 x g fo 30 min at 4º C, neutralized with 1 M of KOH solution and filtrated right through in a 0.22 μm filters.
Samples (20 μL) were injected in a reverse-phase column, Ultrasphere 4.6 mm x 250 mm and 5 μm of particle size (Beckman Coulter, USA) using 0.1 M of potassium
72 phosphate buffer pH 7.0 as a mobile phase at 2.5 mL/min of flow rate. Run was performed isocratically for 20 min. at 30º C and eluent was monitored at 254 nm.
Quantification was made by a standard curve, fitted in a linear regression model made with different nucleotides standard concentrations.
Enzymatic assay
The TMK activity was followed by NADH oxidation, using a coupled enzymatic assay dependent of ADP formation at 37º C (Blondin et al., 1994; Topalis et al., 2005;
Guevara-Hernandez et al., 2012). Reaction mixture contained 50 mM Tris HCl pH 7.4, 50 mM KCl, 5 mM MgCl2, 0.2 mM NADH, 1 mM phosphoenol pyruvate, 1mM DTT, 1 mM of ATP (for standard assay) or variant concentrations and coupling enzymes pyruvate kinase (4U) and lactate dehydrogenase (4U) in a final volume of 1 mL. The reaction was initiated with addition of 0.2 mM of dTMP, dTDP or dUMP, (for standard assay) or variant concentrations of each nucleotide and 5 μg of TMKwssv for 200 seg (protein concentration was determined by BCA method). NADH oxidation was followed by absorption spectroscopy at 340 nm. Activity was expressed in μmol·s-1 and data fitting to a Michaelis-Menten kinetic model was done whit GraphPad Prism 6 software using a non-linear fit model.
Binding affinity of ligands by isothermal titration calorimetry (ITC)
Nucleotide binding was done in a VP-ITC titration microcalorimeter (Microcal Inc., USA). The TMKwssv solution was dialyzed overnight with 25 mM Tris HCL pH 7.4, 150 mM NaCl, 5 mM MgCl2 and 10% ethylene glycol. Substrate solutions were prepared with final dialysis buffer solution. Sample cell (1.4 mL) was filled with
73 TMKwssv solution and reference cell was filled with final dialysis buffer solution. The titrations were made with substrate solution in a 1:20 protein-ligand rate and substrate injections were of 8 μL when saturation was observed. The ITC experiment was performed at 25 ºC and 199 rpm. Data were fitted to a sequential-binding site model by a no-linear regression analysis using Microcal Origin Software to yield binding constant (KB), and enthalpy (ΔH) and entropy (ΔS) changes.
Docking
Molecular modeling of TMKwssv 3D structure was made using MOE 2012 version (Chemical Computing Group) using as template the aminoacid sequence and crystallographic structure of wild type human thymidilate kinase PDB 1E2F (Ostermann et al., 2000). Molecular docking of TMKwssv substrate: dTMP, d4TMP, dTDP and ADP was analyzed.
RESULTS AND DISCUSSION
Identification of a thymidine diphosphate kinase activity on TMKwssv
The monophosphate kinase activity of TMK was reported (Guevara-Hernandez et al., 2012). To further demonstrate the kinase activity, was used a radioactive assay (Neuhard et al., 1965), and a HPLC method (Ryder, 1984) to discriminate between mono, di and tri-phosphorylated nucleotides. For the radioactive assay (Figure 1), the reactions were separated in a thin layer alumina surface and further imaged. Formation of a intense spot was found to be dependent on addition of the TMKwssv as reported for other nucleotides (Van Rompay et al., 1999). That could be [β-P32] dTDP, but in the
74 absence of a known radiolabeled standard, a chromatographic method was used to characterize the formed species.
An HPLC method (Ryder 1985) was used to have an independent method to validate the formation of dTDP. Some viral nucleotide kinases, are able to perform consecutive phosphorylation steps or use different nucleotides as phosphate receptors (Perozzo et al., 2000; Gardberg et al., 2003). In this technique, the nucleotides are separated by polarity, having the triphosphorylated species a smaller retention time compared to the monophosphate nucleotides. After a three-hour reaction the peak for ADP (continuous line) appeared as compared to the blank line (spotted line) and that dTMP was reduced (Figure 2). Although ATP and dTDP peaks overlap, the height decrease as result of the consumption of the ATP, but more interestingly, a peak with a smaller retention time also appeared. Using dTTP as a standard (un-continuous line) the dTTP formation was confirmed and resulted from further phosphorylation of dTDP.
This result lead us to further characterize the kinetics and thermodynamics of nucleotide binding to TMKwssv.
Kinetics studies of TMKwssv
TMKwssv followed the Michaelis-Menten kinetics (Figure 3) for the natural substrate dTMP and the novel substrate dTDP. The phosphorylation of substrate of thymidylate synthase dUMP, was tested to evaluate if it presented thymidine kinase (TK) activity. A coupled enzyme assay was used, and a Michaelis-Menten model was used to calculate the Michaelis constant (Km), the turnover number kcat and the catalytic efficiency kcat/Km. The Km for the cognate substrate dTMP was 0.11 ± 0.014 μM and
75 for the phosphate-donor ATP was 0.092 ± 0.0085 μM. The enzyme was also able to use dTDP as a substrate (Figure 3, panel A), but less efficiently compared to dTMP. The value of Km for dTDP was 0.25 ± 0.022 μM. While dUMP was used even less efficiently and it is likely that all of it is used by TS. The turnover number kcat was 3.56
± 0.12 s-1 for dTMP and 3.16 ± 0.062 s-1 for ATP. These results show a higher catalytic efficiency of TMKwssv natural reaction than other TMK kinases analyzed previously (Table 1). The TK activity of the TMKwssv domain with nucleoside deoxy thymidine
(dT) was tested. No activity was detected towards that substrate on the coupled enzymatic assay (data no shown).
In order to investigate the reaction mechanism of, a series of Lineweaver-Burk plots where done. The dTMP concentration was changed at different concentrations of ATP a pattern indicative of a random sequential mechanism, as previously reported for mouse TMK (Cheng y Prusoff 1973). This mechanism has been reported for others TMK where dTMP is bound first, followed by ATP. However, Streptococuus pneumoniae TMK has a different reaction mechanism ordered bi-bi, in which the TMK-ATP complex occurs first and the a ternary TMK-ATP-dTMP complex is formed towards catalysis (Petit y Koretke 2002).
Binding of substrates to TMKwssv
As mentioned above, the TMKwssv kinetic analysis suggested a sequential reaction mechanism. To study the energetics of binding, TMKwssv was titrated with either dTMP or ATP. A sequential mechanism was clearly supported as dTMP was tightly bound to the enzyme and ATP was not bound(Figure 4).
76 Unlike TMK of Streptococcus pneumonia (Petit y Koretke 2002) TMKwssv was able to bind dTMP and dTDP in complex with AMP-PNP. This type of interaction, was observed before between human TMK dTDP-complexed with ADP, and it assume, that the complex formed is in the direction of the reaction, i.e. just before the release of product (Ostermann et al., 2000), and not as a substrate as with TMKwssv. As a herpes virus TK, TMKwssv only binded ATP when phosphate receptor form protein-nucleotide complex (Perozzo et al., 2000), and is capable to perform sequential phosphorylation steps.
Kinetic and calorimetric assays showed diference between dTMP and dTDP affinities for TMKwssv. Based on these results it is proposed that the double phosphorylation of dTM does not occur consecutively, since on the phosphorylation reaction starting from dTMP, there are very low levels of dTTP detected by HPLC. These results suggest that first dTMP is used as receptor of phosphate and dTDP is used until dTMP is exhausted.
(data not shown)
Union capacity of the nucleoside analogue d4TMP in complex with AMP-PNP is offend use for structural studies of TMK (Osterman, lavie,) Differences in affinity constants were also tested, suggest there may be a difference in the arrangement the binding pocket of tmk between d4TMP and substrate (Figure 7).
CONCLUSION
The reaction mechanism of phosphorilation of dTMP by TMKwssv, was established, it is an ordered bi-bi mechanism that has not been observed in other TMK and the kinetic
77 constants determined for the natural substrates of the enzyme showed a high efficiency of dTDP formation.
The quantitative dTDP and dTTP formation by the TMKwssv domain of WSSV ORF 454.was demonstrated Affinity constants of the TMKwssv substrates were kinetically determined, compared and analyzed with nucleoside analogs. d4TMP affinity was similar to the natural substrate. These results could be very useful to establish treatment strategies, by using drugs which have been used for the treatment of viral diseases
Although it has not yet been demonstrated that the viral kinase TK-TMK gene is separated in vivo in two domains during viral infection, this work has shown that the separation of the two domains is biochemically and structurally feasible and the product of ORF 454 of the white spot virus bifunctional gene is capable of performing all three phosphorylation reactions necessary for the formation of dTTP.
78 FIGURE LIST
79 Figure 1. dTDP formation catalyzed by TMKwssv. Formation of [β32P] dTDP. Spot 1.
Blank, (reaction mixture without TMKwssv). spots 2-5, 40 ng of TMKwssv at 2, 4, 8 y 16 minutes. Spots 6-9, 100 ng of PCNA E. histolitica (negative control) at 2, 4, 8 y 16 min.
80 Figure 2. dTTP formation by TMKwssv. Chromatogram of TMKwssv+dTDP+ATP) reaction. dTTP y ADP formation shown after three hours of reaction. Uncontinue line:
dTTP standar. Continue line: reaction after 3 hours. Spotted line: blank reaction (reaction mixture without TMKwssv).
81 Figure 3. Michaelis-Menten and Lineweaber-Burk plots. A. Nucleotides of dTTP synthesis: de novo and savage pathways. dT (No activity shown), dTMP y dTDP (see text for details). B Double reciprocals plots, for bi-substrate enzymes, shown as a sequential reaction mechanism.
A
B
82 Figure 4. ITC TMKwssv thermograms for dTMP and ATP. A, dTMP titration. B ATP titration, no binding heat was detected.
83 Figure 5. Binding of nucleotide and TMKwssv-AMP-PNP complex thermograms.
A) dTMP Bindig, B) dTDP binding and C) d4TMP binding.
84 Figure 6. TMKwssv tridimensional model. TMKwssv ternary complex with ADP, dTMP y d4TMP, obtained by in silico modeling. The conformation of the catalytic cavity formed by the P-loop, lid loop, and the loop with the catalytic residues is shown.
85 Table 1. Kinetic parameters comparison
TMK
Km(μM) dTMP
Kcat/Km (s-1mM-1) dTMP
Km(μM) ATP
Kcat/Km (s-1mM-1) ATP
Km(μM) dTDP
Kcat/Km dTDP
Cita
WSSV 0.11 31690 0.092 34164 0.2515 3548 This work
E. coli 2.7 5600 8 1875 Lavie et al.
(Lavie y Konrad)
M. tuberculosis 40 1000 100 45 Munier-
Lehmann et al (2001)
H. sapiens (silvestre)
5 140 5 140 Ostermann et
al.
(Ostermann et al., )
Levadura 9 3900 190 184 Lavie et al.
(1998)
P. falciparum 10.7 460 78.6 62 Wititingham
et al. (2010)
B malayi 17 2240 66 577 Doharey et
al. (2014)
S. pneumoniae 66 134 235 38 Petit y
Koretke (2002)
86 Table 2. Thermodynamic parameters and binding constants
* Better adjust to one site
ITC data Nucleotide
dTMP dTDP d4TMP *
Kd1 (mM)
3.28 6.25 3.59
Kd2 (mM)
403 2118
ΔH1cal/mol
-1.35x104 -1.50x104
ΔH2cal/mol
-4.51x104 -1.04x105
ΔS1 cal/mol/deg
-20.4 -26.6 -33.6
ΔS2 cal/mol/deg
-136 -459 -1.74x104
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