J Bioenerg Biomembr (2016) 48:301–308 DOI 10.1007/s10863-016-9660-1

Arginine shows nucleoside diphosphate kinase-like activity toward deoxythymidine diphosphate

Alonso A. Lopez-Zavala1 & Rogerio R. Sotelo-Mundo2 & Jose M. Hernandez-Flores2 & Maria E. Lugo-Sanchez2 & Rocio Sugich-Miranda1 & Karina D. Garcia-Orozco2

Received: 19 October 2015 /Accepted: 1 April 2016 /Published online: 12 April 2016 # Springer Science+Business Media New York 2016

Abstract kinase (AK) (ATP: L-arginine (Arg124, 126 and 309) stabilize the dTDP phosphate groups , E.C. 2.7.3.3) catalyzes the reversible and the pyrimidine base interact with His284 and Ser122. transfer of ATP γ-phosphate group to L-arginine to synthetize These results suggest that LvAK bind and phosphorylate phospho-arginine as a high-energy storage. Previous studies dTDP being ATP the phosphate donor, thus describing a novel suggest additional roles for AK in cellular processes. Since alternate nucleoside diphosphate kinase-like activity for this AK is found only in invertebrates and it is homologous to . from vertebrates, the objective of this work was to demonstrate nucleoside diphosphate kinase-like activ- Keywords Arginine kinase . Litopenaeus vannamei . dTDP . ity for shrimp AK. For this, AK from marine shrimp Nucleoside diphosphate kinase . ITC . Tryptophan Litopenaeus vannamei (LvAK) was purified and its activity quenching . Docking was assayed for phosphorylation of TDP using ATP as phos- phate donor. Moreover, by using high-pressure liquid chroma- tography (HPLC) the phosphate transfer reaction was follow- Introduction ed. Also, LvAK tryptophan fluorescence emission changes were detected by dTDP titration, suggesting that the hydro- Arginine kinase (AK) (ATP:L-arginine phosphotransferase, phobic environment of Trp 221, which is located in the top of E.C. 2.7.3.3) is an enzyme that catalyzes the reversible trans- the , is perturbed upon dTDP binding. The kinetic fer of high energy ATP γ-phosphate group to L-arginine. AK constants for both substrates Arg and dTDP were calculated belongs to a wide family of guanidine by isothermal titration calorimetry (ITC). Besides, docking that use creatine, glicocyamine, arginine, taurocyamine, calculations suggested that dTDP could bind LvAK in the ofeline and lombricine, thus playing a key role in the produc- same cavity where ATP bind, and LvAK basic residues tion and use of energy (Suzuki et al. 1997). When high ATP levels are present in the cell, AK phosphorylates L-Arg and when a burst of energy is required, phospho-L-Arg phosphor- Electronic supplementary material The online version of this article (doi:10.1007/s10863-016-9660-1) contains supplementary material, ylates ADP to restore cellular energy (Ellington 2001). This which is available to authorized users. enzyme has a function similar to creatine kinase in vertebrates (Wallimann et al. 1998). AK is only found in invertebrates and * Karina D. Garcia-Orozco it has been isolated from several sources as a monomeric [email protected] 40 kDa protein; however, in some cases it has been purified as an homodimer of 80 kDa (Guo et al. 2003; Shi et al. 2012; 1 Departamento de Ciencias Químico Biológicas, Universidad de Wu et al. 2010). Biochemical and kinetic evidence indicates Sonora, Calle Rosales y Blvd. Luis Encinas s/n, Col. Centro, Hermosillo, Sonora 83000, México that AK catalytic mechanism consists of a reversible in-line phosphate transfer between ATP and the L-Arg-guanidine 2 Laboratory, Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD), Carretera a Ejido La group (Ellington 2001). Victoria Km 0.6, Apartado Postal 1735, Hermosillo, Sonora 83304, The role of AK in energy metabolism of marine México crustaceans is well documented (France et al. 1997; Wang 302 J Bioenerg Biomembr (2016) 48:301–308 et al. 2009; Yao et al. 2009) and it is one of the major allergens linear gradient from 0 to 1 M NaCl in buffer A. The fractions in shrimp (Garcia-Orozco et al. 2007; Yao et al. 2009). Also, obtained were analyzed by 12 % SDS-PAGE. Gels were crystallographic studies in L. vannamei AK (LvAK) have stained with silver nitrate as purity criteria. The pure fractions shown that it has a similar fold to other invertebrate AK and containing the protein were pooled and stored at 4 °C until use. the active site is conserved to bind both nucleotide and phosphagen substrates (Lopez-Zavala et al. 2013; Lopez- LvAK enzymatic activity assay Zavala et al. 2012). LvAK nucleotide is com- prised by basic amino acids that stabilize negative charges of Phospho-transfer enzymatic activity was measured by a ATP-phosphate groups (Lopez-Zavala et al. 2013). Likewise, coupled assay based on the oxidation of NADH (Blethen Trp221 is located in the top of its active site and the hydro- 1972). In the first reaction, LvAK uses ATP-Mg+2 as the phos- phobic environment is altered during the interaction of an phate donor to L-arginine leading to P-Arg and ADP-Mg+2.In inhibitor or other compounds with AK (Wang et al. 2011; the second reaction, the newly synthesized ADP-Mg+2 is re- Wu et al. 2009). generated to ATP-Mg+2 by the action of (PK) The importance of the phosphagens such as phosphocrea- in the presence of phosphoenol pyruvate. Finally, the third tine in the muscle during exercise in vertebrate organisms has reaction is the pyruvate reduction to lactate using lactate de- been well documented (Ellington and Hines 1991; France et hydrogenase (LDH) and NADH as reducing agent. The al. 1997; Jubrias et al. 2001; Vishnudas and Vigoreaux 2006). NADH oxidation rate was measured spectrophotometrically Several studies have shown differential AK expression in mus- at 340 nm. One unit is the amount of enzyme that converts 1.0 cle of shrimp subjected to white spot and Taura viral infection μmole of L-arginine and ATP to phospho-L-arginine and (Ma et al. 2014; Rattanarojpong et al. 2007;Wangetal.2006) ADP per minute at pH 8.6 and 30 °C. Control reactions con- or hypoxia (Abe et al. 2007; Jiang et al. 2009). This evidence tain all reactants except the enzyme and were monitored until suggests that AK is involved in multiple physiological pro- no absorbance changes were detected (Blethen 1970). cesses and probably AK has other cellular functions besides Substrates L-arginine, dADP, dTDP, dCDP, dGDP, dTMP bioenergetics.. Therefore, the capacity of LvAK to phosphor- and thymidine were tested as phosphate acceptors in the assay ylate nucleotides using ATP as phosphate donor was evaluat- described above. The conditions of the assay have been de- ed; this is to have a Bmoonlighting^ activity where one poly- scribed elsewhere (Blethen 1972). The final concentration of peptide can have two or more functions (Jeffery 2003). L-arginine and all diphosphate deoxy-nucleotides was 17 mM in the assay cuvette. Determination of kinetic constants by isothermal titration Materials and methods calorimetry (ITC). ITC experiments were performed using a titration isother- Shrimp AK Purification (LvAK) mal micro calorimeter VP-ITC (Microcal Inc., Northampton, MA, USA) in kinetic mode. The reference cell was filled with All chemical and reagents used were from Sigma-Aldrich, fresh dialysis buffer C (100 mM glycine buffer pH 8.6, (Toluca, México), they were reagent grade or better. LvAK 1.4 mM magnesium chloride and 10 mM 2-mercaptoethanol). was purified according to a previously reported method LvAK was extensively dialyzed overnight against buffer C (Garcia-Orozco et al. 2007). Briefly, 5 g from Pacific whiteleg prior to the experiment. The solutions 1 mM ATP, shrimp Litopenaeus vannamei tail muscle were homogenized 5 mM arginine or 5 mM dTDP were prepared with buffer C. with ice-cold 50 ml of extraction buffer (0.1 M Tris.HCl The cell was filled with 1 mM solution of the enzyme LvAK pH 8.0, 1 mM EDTA, 5 mM NaN3, 25 mM PMSF, 10 mM with 1.4 mM of MgCl2 as and titrated to separate with 2-mercaptoethanol) using a Polytron homogenizer (Glenn solutions of 1 mM ATP, 5 mM arginine or 5 mM dTDP at a Mills Co., Clifton, NJ, USA) at 30,000 RPM, 3 pulses of temperature of 30 °C. Injections were of 10 μlspacedevery 10 s each in an ice bath. The homogenate was stirred for 800 s to ensure complete equilibrium. Subsequently, the data 16 h at 4 °C and clarified at 12,000 × g for 30 min at 4 °C. was processed using the program Microcal Origin®, adjusted The supernatant was subjected to serial ammonium sulfate to a single site kinetic model by a nonlinear regression analy- precipitation at 70 and 90 % of saturation. The pellet was sis to obtain the Michaelis-Menten constants for this enzyme. dissolved in 5 ml of buffer A (10 mM Tris.HCl pH 8.0, 0.1 mM EDTA, 10 mM 2-mercaptoethanol) and extensively Identification by HPLC of the LvAK reaction products dialyzed against buffer A using a 10 kDa-cutoff cellulose membrane tubing. To monitor the formation of the LvAK reaction products, two Dialyzed protein was loaded into a 5 ml column of Q- different methodologies were used in a high-performance liq- Sepharose Hi-Trap (GE Healthcare, USA) previously equili- uid chromatograph model 1100 (HPCL, Hewlett Packard Inc., brated with buffer A. The bound protein was eluted with a USA). First, phospho-L-arginine (P-Arg) was quantified as J Bioenerg Biomembr (2016) 48:301–308 303 previously described (Viant et al. 2001). The samples were nucleotide-binding site of LvAK crystal structure; all ligands treated with 7.5 % trichloroacetic acid to precipitate all the were previously removed from the active site. Nucleotide po- protein present. Subsequently, the supernatants obtained and sitioning was done by the Alpha Triangle method and the standard solutions at a concentration of 1 mM ATP, arginine London (dG) scoring function for at very least 80,000 poses. and P-Arg, underwent an isocratic run of 2.0 ml/min in mono- After, 30 independent poses were refined by 500 iterations basic phosphate buffer 20 mM pH 2.6 and acetonitrile at a with the Force field scheme and the Affinity dG rescoring 72:28 ratio in a reverse phase C18 column (100 × 4.6 mm., function under the CHARMM27 force field. Structural visu- 3 μm particle size). ATP standard compound and the forma- alizations and figures were performed using CCP4 molecular tion of P-Arg were identified with a UV–VIS detector at graphics program (McNicholas et al. 2011). 205 nm. Also, the formation of dTTP was detected as previously described (Ryder 1985). Enzymatic reactions were stopped Results and proteins were removed by precipitation with 0.6 mM

HClO4 at 0 °C. Subsequently, the acid was neutralized with Shrimps AK is able to phosphorylate dTDP using ATP 1 M KOH, and all salts and precipitates were removed by as phosphate donor filtration. The samples and standards were run isocratically with potassium phosphate buffer (40 mM KH2PO4, The AK activity with arginine as the substrate was previously 60 mM M K2HPO4 pH 7.0) on a reversed phase column reported (Garcia-Orozco et al. 2007). In this study, using an μBondapak C18 (3.9 mm ID × 30 cm, 5 μm particle size) enzymatic-coupled assay LvAK presented an activity of 7.4 and detected by UV–VIS at 254 nm. The signals were quali- U/mg of protein that is comparable to specific AK activities of tatively identified by their retention times, superposition of other invertebrates like 3.0 U/mg for the beetle (Cissites standards and samples signals, and also by appearance or dis- cephalotes) and 17.0 U/mg for the lobster (Homarus vulgaris) appearance of recognized signals from various components of (Tanaka et al. 2007). To test whether LvAK has NDK-like each reaction. enzymatic activity, dTDP, dCDP, dADP and dGDP were test- ed as phosphate acceptors and ATP as the phosphate donor. Determination of dissociation constant of dTDP to LvAK Only dTDP led to NADH oxidation, LvAK did not phosphor- by tryptophan quenching fluorescence ylate the other nucleotides. Interestingly, among all the non- cognate substrates tested (dADP, dTDP, dCDP, dGDP, dTMP This method was used in order to calculate the dissociation and thymidine), LvAK was only able to phosphorylate dTDP constant (Kd) of LvAK to dTDP. The emission spectra were with an activity of 1.43 U/mg protein, about 20 % of enzyme collected in a spectrofluorometer model QM-2003 (Photon activity with the cognatel substrate L-arginine. Technology International, Inc., USA) with a 75 W Xenon lamp as light source. The samples were excited at 295 nm Kinetic characterization of LvAK by isothermal titration and Trp emission was collected from 300 to 400 nm. Protein calorimetry (ITC) concentration of LvAK was 1 μM and titrated with aliquots of 3 μl of a 50 mM dTDP stock solution. Sample was equilibrat- Measuring enzymatic parameters for require either ed before fluorescence reading. Inner filters effects were neg- radioactive methods or coupled enzymatic assays where ligible and calculations were made as previously reported theuseofATPiscoupledtotheoxidationofNADHor (Arvizu-Flores et al. 2009). ΔF was calculated subtracting NADPH. Isothermal titration calorimetry (ITC) is a tech- the AK fluorescence intensity emitted at 333 nm to the fluo- nique traditionally used to measure binding affinity con- rescence in the presence of dTDP. Average ΔFfromtriplicate stants of substrates or ligands to or receptors. experiments was plotted against dTDP and adjusted to a hy- However, mathematical models have been developed to perbolic non-linear model using GraphPad. correlate the energy gained or generated during an enzy- matic reaction and obtaining the Michaelis-Menten kinetic Molecular docking of TDP in the LvAK constants (Freire et al. 1990; Morin and Freire 1991; nucleotide-binding site Williams and Toone 1993). The LvAK Michaelis-Menten constant obtained for ATP Crystal structure of LvAK in a dead-end ternary state complex (Km = 0.0986 mM, Fig. 1 Panel A) indicated that it has a with ADP-Mg+2, arginine and nitrate ion was used as model higher affinity to the active site compared to L-arginine for docking experiments (PDB: 4BG4) (Lopez-Zavala et al. (Km = 0.322 mM, Fig. 1 Panel B). The novel substrate iden- 2013). TDP docking was performed with the software MOE tified in this work was dTDP, obtaining a value of ver. 2013.08 using the Induced-Fit protocol (Chemical Km = 0.653 mM (Fig. 1 Panel C), in the same order of mag- Computing Group, Canada). The dTDP was docked in the nitude of the cognate substrate. These kinetic parameters are 304 J Bioenerg Biomembr (2016) 48:301–308

Fig. 1 Kinetic calculations using isothermal titration calorimetry (ITC). 1 mM ATP in the syringe. Panel B) Titration of LvAK with 5 mM Experimental thermograms were obtained by ITC in kinetic mode and arginine. Panel C) Titration of LvAK with 5 mM dTDP. Experimental data adjusted in Origin® software. Panel A) Titration of LvAK with details are described in BMaterials and methods^ similar to other arginine kinases (Table 1). In most cases these used were ATP, dTDP, dTTP, arginine and P-Arg at a concen- enzymes have higher affinity for ATP, except oyster AK that tration of 1 mM. has more affinity for arginine (Fujimoto et al. 2005). When LvAK reacteded with ATP and the cognate substrate Comparing the Km values for dTDP and arginine, these are arginine, a decrease in the peaks corresponding to arginine (re- consistent with better activity towards L-Arg compared to tention time 2.5 min) and for ATP (retention time 3.2 min) were dTDP. observed, whereas for phospho-arginine (P-Arg) there was a dramatic increase in the presence of the (Fig. 2 panel A). The control experiments shown as a dashed line correspond Monitoring formation of reaction products by HPLC to a mixture of Arg, ATP and P-Arg without enzyme. More interestingly, when ATP and dTDP was incubated with To have an independent confirmation that the activity and the LvAK, the peak corresponding to dTDP and ATP decreased reaction heats obtained by ITC represent the phosphorylation significantly and dTTP appeared as well as ADP (Fig. 2 Panel of dTDP, we used reverse phase HPLC to identify the di and B). These biochemical results suggest that LvAK could have a tri- phosphorylated forms of deoxy thymidine. The standards moonlighting function as NDK-like activity.

Table 1 Michaelis-Menten constants for invertebrate arginine Km Arg Km ATP K m dTDP kinases Pacific whiteleg shrimp 0.322 ± 0.043 0.099 ± 0.0037 0.653 ± 0.060 Litopenaeus vannamei (this work) Cockroach 0.49 0.14 – P. americana Brown and Grossman (2004) Hornworm 0.5 2.5 – M. sexta Rosenthal et al. (1977) Oyster 0.35 0.97 – C. virginica Fujimoto et al. (2005)

All values are in mM J Bioenerg Biomembr (2016) 48:301–308 305

Fig. 2 HPLC chromatograms of shrimp AK reactions. Acid-soluble and reaction with LvAK are represented as a solid line. Panel A)LvAK LvAK products were separated by C18 reverse phase chromatography; reaction when arginine is used as a substrate. Panel B) LvAK reaction control reactions (without enzyme LvAK) are represented as a dotted line products when dTDP is used as a substrate

Nucleotide binding to LvAK by Trp fluorescence emission fluorescence quenching experiments. Sequence analysis of LvAK showed a well-conserved tryptophan residue (Trp221 In this work, fluorescence emission was used to measure nu- in LvAK) among other invertebrates. Trp221 is located in the cleotide binding to LvAK. Because dTDP was the only sub- top of active site and its hydrophobic environment is perturbed strate for LvAK, the other nucleotides were not tested in the upon binding of several ligands to AK as the natural substrate Trp fluorescence experiments. Trp fluorescence emission ATP (Guo et al. 2004) and other compounds such as flavo- quenching (333 nm) due to dTDP addition to LvAK was noids (Wang et al. 2011). The results obtained by docking observed, since the fluorescence intensity decreased upon li- experiments showed that dTDP adopt a well-ordered confor- gand titration (Fig. 3 inset). The data was adjusted to a single mation in the ADP-Mg+2 binding site (Fig. 4). Trp221 oc- binding site (R2 = 0.997) as previously described, finding a cupies a similar position when dTDP was docked in the dissociation constant for dTDP Kd = 0.520 ± 0.097 mM LvAK active site as ADP-Mg+2 obtained by X-ray crystallog- (Fig. 3). This value is in the same order of magnitude of the raphy (Lopez-Zavala et al. 2013). ITC-calculated Michaelis-Menten constant Km = 0.653 In detail, the pyrimidine base stacks in the hydrophobic ± 0.06 mM. Binding of the cognate substrate L-Arg did not pocket formed between His284, Met233, and the carbon cause a change in the fluorescence emission spectra (data not atoms (CB, CD and CE) from Arg124 lateral chain of LvAK shown). (Fig. 5). Also, the base amide group and oxygen makes a hydrogen bond with Ser122 carboxylate and Asp324, respec- tively. In the same way, a network of ionic interactions was dTDP binding site by molecular docking found between the phosphates group and arginine residues Molecular docking was done to gain an structural insight into the interaction of dTDP and LvAK active site observed in Trp

Fig. 4 Surface representation of dTDP docked into the LvAK active site. ADP-Mg+2 and nitrate ion correspond to the LvAK crystal structure from Fig. 3 Trp quenching titration with dTDP. Fluorescence Trp emission (PDB code: 4BG4). Trp221 is located at the top of nucleotide binding spectra were recorded and data was adjusted to a nonlinear regression site. Surface is colored by electrostatic potential gradient (blue represents model. The insert figure shows the fluorescence emission spectra at positive and red negative charges). Both dTDP and ADP are shown as different concentrations of the substrate (dTDP), with a peak at a cylinders colored in yellow and green, respectively; Mg+2 (brown)and wavelength of 333 nm nitrate ions (atom type) are shown in ball and stick representation 306 J Bioenerg Biomembr (2016) 48:301–308

In this work, enzymatic assays and the identification of dTTP formation by HPLC is presented as evidence that shrimp AK has dTDP-nucleoside diphosphate kinase-like ac- tivity. The results obtained suggest that LvAK besides having a main role in bioenergetics may have another function cata- lyzing the third phosphorylation of deoxy-thymine diphos- phate as a nucleoside diphosphate kinase (Quintero-Reyes et al. 2012). This broad kinase activity was also reported for E. coli (ADK) (Lu and Inouye 1996; Willemoes and Kilstrup 2005). E. coli NDK-null mutants were able to synthetize DNA precursors by other pathways (ADK by-pass) to support the same viability as the wild-type strain (Lu and Inouye 1996; Willemoes and Kilstrup 2005). Trp quenching upon dTDP binding suggests that the en- Fig. 5 Molecular interactions between dTDP and LvAK in the nucleotide-binding. Substrates of ternary analog complex (ADP-Mg+2, zyme has a Trp near the nucleotide-binding site. LvAK se- L-arginine and NO3) of LvAK crystal structure (PDB code: 4BG4) quence has two Trp residues and crystallographic data shows were superposed as reference. Residues that make interaction with that Trp221 is located at the top of the active site (Janin et al. dTDP are shown as cylinders colored by atom type. Non-covalent 2000;Lopez-Zavalaetal.2013). Trp fluorescence quenching interactions (H-bond and saline bridges) are represented as black dotted line. Docked dTDP is shown as cylinders colored by atom type with is well documented in AK upon binding of ATP in the ternary carbons atoms in yellow analog complex with L-Arg and nitrate ion (Guo et al. 2004; Liu et al. 2011), flavonoids (Wang et al. 2011;Wuetal.2009), gallotannin (Adeyemi et al. 2014), creatine kinase (Trp227) (Arg124, 280 and 309). Trp221 is place in a very close con- and pyruvate kinase (Trp157) (Wennefors et al. 2008) and formation to TDP, also as noted in experimental data from silver/gold nanoparticles (Adeyemi and Whiteley 2014). LvAK in complex with ADP-Mg+2 and arginine. ATP and dTDP have similar physicochemical properties and it is possible that both substrates bind to the same cavity. Other kinases, such as nucleoside diphosphate kinase interact with Discussion both acceptor and donor phosphate substrate in the same bind- ing site (Janin et al. 2000). Our results suggest that LvAK One gene, one enzyme, one function is a paradigm long gone binds dTDP in the same binding site as for ATP. (Jeffery 2003). More evidence appears that redundancy exists in The theoretical molecular model provides structural infor- protein function and LvAK appears to be the case. Both ribo- mation that explain the changes in fluorescence quenching and deoxynucleotides must be phosphorylated by several intra- observed in LvAK during the interaction with TDP. Also, it cellular kinases before they are incorporated by suggest that LvAK could bind both the phosphate donor during cell duplication or other nucleic acid repair mechanism (ATP) and the acceptor (TDP) in the same binding site as (Mathews 2014). The third phosphorylation is a key step cata- reported for other nucleotide kinases (creatine kinase, pyru- lyzed by nucleotide diphosphate kinase (NDK) that can phos- vate kinase and 3-phosphoglicerate kinase) that show sub- phorylate both deoxy and ribonucleotides (Gonin et al. 1999). strate promiscuity and catalyze the third nucleotide phos- NDK shows high substrate promiscuity and can phosphorylate phorylation step (Janin et al. 2000; Lascu et al. 2000; pro-drugs to the active form (Schaertl et al. 1998). Nevertheless, Varga et al. 2011). NDK has low activity towards substrates analogues lacking the Nucleoside diphosphate kinase (NDK) can use both purine ribose 3′-hydroxil group (Schaertl et al. 1998). and pyrimidine nucleotides in the same biding site with min- Other kinases as creatine kinase (CK), pyruvate kinase and imal conformational changes (Janin et al. 2000; Williams and 3- can also catalyze the third phos- Toone 1993). Therefore, we hypothesize that LvAK could phorylation step (Deville-Bonne et al. 2010). Human creatine bind both the phosphate acceptor as well as the donor like kinase has been found to be responsible for the activation of NDK. Further kinetics and structural studies are needed to two anti-HIV drugs (ddCDP and ddADP), which could not be elucidate the mechanistic behavior of this reaction catalyzed phosphorylated by cytosolic NDK (Krishnan et al. 2002; by LvAK. Wennefors et al. 2008). The findings that other cellular kinases Transcriptomic studies have shown that under a viral chal- with relative high substrate promiscuity could have other met- lenge LvAK expression is increased, suggesting that it may abolic roles besides to its classical cellular function are well have a role in nucleotide metabolism. The nucleotide triphos- documented (Arvizu-Flores et al. 2009; Guevara-Hernandez phate pool for DNA replication is maintained by several ki- et al. 2012;Nelsonetal.2008; Quintero-Reyes et al. 2012). nases and those are up regulated during viral infections. To our J Bioenerg Biomembr (2016) 48:301–308 307 knowledge, the other example of this broad kinase activity is Garcia-Orozco KD, Aispuro-Hernandez E, Yepiz-Plascencia G, the E. coli adenylate kinase (ADK), which can also work as Calderon-De-La-Barca AM, Sotelo-Mundo RR (2007) Int Arch Allergy Immunol 144:23–28 nucleoside diphosphate kinase (Lu and Inouye 1996; Gonin P, Xu Y, Milon L, Dabernat S, Morr M, Kumar R, Lacombe ML, Willemoes and Kilstrup 2005). Lu and Inouye noticed that Janin J, Lascu I (1999) Biochemistry 38:7265–7272 NDK null mutants were viable and that were rescued by the Guevara-Hernandez E, Garcia-Orozco KD, Sotelo-Mundo RR (2012) – ADK. These structural approaches suggest that LvAK could ProteinPeptLett19:1220 1224 Guo SY, Guo Z, Guo Q, Chen BY, Wang XC (2003) Protein Expr Purif bind and phosphorylate dTDP in the same cavity for ATP that 29:230–234 is well documented in NDK (Janin et al. 2000). Guo Q, Zhao F, Guo SY, Wang X (2004) Biochimie 86:379–386 Janin J, Dumas C, Morera S, Xu Y, Meyer P, Chiadmi M, Cherfils J (2000) J Bioenerg Biomembr 32:215–225 Jeffery CJ (2003) Trends Genet 19:415–417 Conclusion Jiang H, Li F, Xie Y, Huang B, Zhang J, Zhang J, Zhang C, Li S, Xiang J (2009) Proteomics 9:3353–3367 LvAK is a key phosphagen for invertebrate bioenergetics and Jubrias SA, Esselman PC, Price LB, Cress ME, Conley KE (2001) J Appl – this work show that this enzyme is able to phosphorylate Physiol 90:1663 1670 Krishnan P, Fu Q, Lam W, Liou JY, Dutschman G, Cheng YC (2002) J dTDP. In this paper, we provide biochemical evidence using Biol Chem 277:5453–5459 an enzymatic coupled assay, ITC and HPLC to propose that Lascu L, Giartosio A, Ransac S, Erent M (2000) J Bioenerg Biomembr shrimp AK has NDK-like activity producing dTTP. Also, 32:227–236 – fluorescence analysis and docking calculation suggest that Liu N, Wang JS, Wang WD, Pan JC (2011) Int J Biol Macromol 49:98 102 dTDP make similar interactions with the LvAK active site as Lopez-Zavala AA, Sotelo-Mundo RR, Garcia-Orozco KD, Isac-Martinez reported for ADP in the crystal structure of LvAK. F, Brieba LG, Rudino-Pinera E (2012) Acta Crystallogr Sect F: Mechanistic questions regarding this novel activity could be Struct Biol Cryst Commun 68:783–785 addressed using structural methods, considering that LvAK is Lopez-Zavala AA, Garcia-Orozco KD, Carrasco-Miranda JS, Sugich- Miranda R, Velazquez-Contreras EF, Criscitiello MF, Brieba LG, amenable for crystallographic studies Rudino-Pinera E, Sotelo-Mundo RR (2013) J Bioenerg Biomembr 45:511–518 Acknowledgments Jose Max Hernández-Flores was supported by a Lu Q, Inouye M (1996) Proc Natl Acad Sci U S A 93:5720–5725 student fellowship from CONACyT (Mexico’s National Science and Ma FF, Liu QH, Guan GK, Li C, Huang J (2014) Gene 539:99–106 Research Council). Alonso A. Lopez-Zavala was supported by Mathews CK (2014) FASEB J 28:3832–3840 CONACyT under the grant BFondo Consolidacion Institucional (I0007- Mcnicholas S, Potterton E, Wilson KS, Noble ME (2011) Acta 2015-01, #250973)^. 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