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Article

The Arginine Kinase from the Tick Rhipicephalus sanguineus Is an Efficient Biocatalyst

by
Ana C. Gomez-Yanes
1,
Elena N. Moreno-Cordova
1,
Karina D. Garcia-Orozco
1,
Aldana Laino
2,
Maria A. Islas-Osuna
3,
Alonso A. Lopez-Zavala
4,
Jesus G. Valenzuela
5 and
Rogerio R. Sotelo-Mundo
1,*
1
Laboratorio de Estructura Biomolecular, Centro de Investigación en Alimentación y Desarrollo, A.C., Carretera Gustavo Enrique Astiazarán Rosas Núm. 46, Ejido a La Victoria, Hermosillo 83304, Sonora, Mexico
2
Instituto de Investigaciones Bioquímicas de La Plata “Dr. Prof. Rodolfo R. Brenner” (INIBIOLP), Centro Cientifico Tecnologico-La Plata CONICET-Universidad Nacional de La Plata, Calle 60 y 120, La Plata 1900, Argentina
3
Laboratorio de Genética y Biología Molecular de Plantas, Centro de Investigación en Alimentación y Desarrollo, A.C., Carretera Gustavo Enrique Astiazarán Rosas Núm. 46, Ejido a La Victoria, Hermosillo 83304, Sonora, Mexico
4
Departamento de Ciencias Quimico Biológicas, Universidad de Sonora, Blvd. Rosales y Bvd. Luis Encinas s/n., Hermosillo 83000, Sonora, Mexico
5
Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, 5601 Fishers Ln, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA
*
Author to whom correspondence should be addressed.
Catalysts 2022, 12(10), 1178; https://0-doi-org.brum.beds.ac.uk/10.3390/catal12101178
Submission received: 23 August 2022 / Revised: 29 September 2022 / Accepted: 30 September 2022 / Published: 6 October 2022
(This article belongs to the Section Biocatalysis)
Browse Figures
Versions Notes

Abstract

:
Arginine kinase (AK) is a reversible enzyme that regulates invertebrates’ phosphagen arginine phosphate levels. AK also elicits an immune response in humans, and it is a major food allergen in crustacea and may be a target for novel antiparasitic drugs. Although AK has been primarily described in the shrimp, it is also present in other invertebrates, such as the brown tick Rhipicephalus sanguineus (Rs), the vector for Rocky Mountain Spotted Fever. Here we report the enzymatic activity and the crystal structure of AK from Rhipicephalus sanguineus (RsAK) in an open conformation without substrate or ligands and a theoretical structure of RsAK modeled bound with the substrate/product (Arg-ADP) in a closed conformation. The Michaelis-Menten kinetics confirmed that RsAK is an efficient biocatalyst due to its high kcat/Km parameter. The recombinant enzyme was expressed in bacteria and purified to a 20 mg/L culture yield. AK is an essential enzyme in invertebrates. Future work will be focused on the RsAK enzymatic inhibition that may lead to novel strategies to control this pest, a burden to animal and human health.

1. Introduction

Energy maintenance requires replenishing adenosine triphosphate (ATP) levels during energy deficits such as sudden muscle contraction and hypoxia. Kinase enzymes temporally supply ATP; in vertebrates is creatine kinase (CK), while in invertebrates is arginine kinase (AK) [1]. Insect flight [2] or escape responses in shrimps [3] require bouts of muscular contraction and efficient kinase biocatalysts to produce ATP from phosphorylated molecules known as phosphagens [4]. Arginine kinases (AK) are also studied in crustacea as an allergen or as the cockroach AK is an elicitor of asthma [5].
The brown dog tick (Rhipicephalus sanguineus) is an invertebrate vector of Rickettsia rickettsii, the causative agent of the Rocky Mountain Spotted Fever or rickettsiosis. It is a re-emerging disease in Mexico with an increased mortality rate in adolescents and children [6,7,8]. One of the biggest challenges when dealing with this disease is that its symptoms are very similar to many other clinical conditions. In addition, climate change affects the distribution and abundance of the vector, the transmission of rickettsia, and the need for novel control strategies, such as RNA interference over AK to inhibit parasite development [9].
Invertebrates use phosphoarginine as their phosphagen, and AK (EC 2.7.3.2) reversible activity keeps the ATP pools in the invertebrate muscle [10] and the nervous system [11]. Biochemically, AK catalyzes reversible phosphoryl transfer between phosphoarginine to adenosine diphosphate (ADP), generating adenosine triphosphate (ATP) and arginine [12]. Although the catalytic-induced conformational change is transient, it can be stabilized using the reaction products and ions. This complex in the closed conformation has been called the transition state analog complex (TSAC), and it was first described in the horseshoe crab AK [13,14]. This work will refer to this as the substrate/product (Arg-ADP) closed conformation.
Antigenic activity has been reported in the AK of some species like Litopenaeus vannamei, Periplaneta americana, Callinectes bellicosus, and Bombyx mori [15,16,17,18]. In addition, crystal structures of AKs from invertebrates such as horseshoe crab Limulus polyphemus [13], sea anemone Anthopleura japonica [19], marine shrimp Litopenaeus vannamei [20], sea cucumber Apostichopus japonicus [21], south American spider Polybetes pythagoricus [22], mud crab Scylla paramamosain [23], among others, have been determined. However, there are no reports of structural studies of ectoparasite’s AKs, which can contribute to the knowledge of the function of this phosphagen kinase in organisms of this kind of parasite. Furthermore, structural studies of the RsAK could reveal species-specific epitopes as biomarkers of tick exposure, which are essential to seeking populations at risk of contracting rickettsiosis.
Herein, we identified the amino acid sequence of RsAK from a transcriptome and genome of the brown dog tick R. sanguineus [24,25], produced RsAK recombinant protein, and determined its crystal structure and its Michaelis-Menten kinetics, showing that it is an efficient biocatalyst for rapid ATP production.

2. Results

2.1. Production of Recombinant RsAK

RsAK (~42 kDa) was obtained in the soluble fraction after the induction with 0.2 mM of IPTG and gradually increased its concentration with time, reaching its maximum production at 18 h post-induction. RsAK was isolated at 40% imidazole from the IMAC chromatography (Figure S1, peak 2 and lanes 5–7 from SDS-PAGE). Size exclusion chromatography (SEC) confirmed the peak corresponding to RsAK has a molecular weight of 42 kDa, ensuring that the RsAK native state is a monomer in solution (Figure S2). This is consistent with most AKs ranging from 36 to 43 kDa [26]. The yield of RsAK was ~20 mg of RsAK per liter of culture after the protein was purified by SEC.

2.2. RsAK Enzymatic Activity

The specific enzymatic activity of the RsAK was 1.5 U/mg, which is in the order of magnitude of the L. vannamei AK with 8.8 U/mg [15]. Other AKs exhibit higher specific activities, such as the spider P. pythagoricus, with a specific activity of 14.4 U/mg [27], Pholucus phalangoides with 15.3 U/mg, Paralithodes kamtschatica with 17.8 U/mg, Jasus lalandii with 24.7 U/mg, Dugesiella hentzi with 58.5 U/mg, and Melanopus bruneri with 15.1 U/mg [26]. The variations in the specific activity may be due to the difference in the different species’ metabolic rates, besides variations in the techniques. Whereas for the Michaelis-Menten parameters for arginine and ATP, RsAK had a higher affinity (lower Km), reflected in excellent catalytic efficiencies for invertebrate AKs (Figure 1, Table 1).

2.3. RsAK Crystal Structure

Crystal structure of the apo-RsAK in its open conformation was obtained at a 1.53 Å resolution. A summary of diffraction and refinement data is given in Table 2. RsAK folds into two domains, the N-terminal one with approximately 100 residues, an α-helix, and the catalytic domain, including the C-terminal region of an eight-stranded antiparallel β-sheet flanked by seven α-helices [23] (Figure 1A). The apo AK structures are superimposable to the Arg-bound experimental crystal structures and are described as the “open” conformation in comparison to the substrate/product bound (or TSAC) models [20].
The electron density for the active site was well defined in the RsAK structure (Figure 2B). The binding loop of the substrate carboxylate group, comprised of residues Asp62, Ser63, Gly64, and Val65, is critical for arginine binding in a lock-and-key mechanism [28]. We also identified the coordinates for the residues interacting with the arginine-guanidinium group binding sites (Glu225, Cys271, and Thr273) (Figure 2C).
After repeated unsuccessful crystallization trials using kits Crystal Screen 1, 2, and Index (Hampton Research) to obtain an experimental model of the closed conformation complex (AK-Arg-NO3-ADP), we modeled RsAK in the TSAC-product conformation. The template used to obtain such a model was the shrimp ternary complex PDB 4BG4 [31]. After alignment of the Cα backbones of the crystallographic apo RsAK (PDB 7RE6) and the RsAK substrate/product (Arg-ADP) closed conformer, we obtained a root mean standard deviation (RMSD) of 1.48 Å (Figure 3).

2.4. Prediction of RsAK Discontinuous Epitopes

This work predicted RsAK discontinuous epitopes using the Disco Tope algorithm (https://services.healthtech.dtu.dk/service.php?DiscoTope-2.0 accessed on 22 August 2022) [32]. It uses a combination of amino acid statistics, spatial information, and exposure surface, using a compilation of discontinuous epitope data from 76 antigen-antibody complex crystallographic structures [32]. Also, it probes the protein structure’s carbon skeleton within a 10 Å sphere, adding the propensity score of the residues in the sphere and subtracting the number of amino acid residues within the sphere [33]. This algorithm found 57 probable antigenic residues in the RsAK, 29 for the L. vannamei AK, 25 for P. pythagoricus, and 23 for L. polyphemus, being RsAK the most antigenic of those AK analyzed.
The AK protein sequences were compared by multiple sequence alignment. The predicted epitopes for the studied species were mapped into the sequence and corresponding 3D structure to visualize the conserved epitopes between the different AKs using PYMOL [34] (Figure 4). The AKs of the five species (R. sanguineus, L. vannamei, P. pythagoricus, S. paramamosain, and L. polyphemus) presented antigenic sites at residues 171–183. In addition, AKs had conserved epitopes at the residues 92–101, but we found four potential RsAK-specific epitopes at residues 157–169, 240–241, 257–258, and 335–341 (Figure 5).

3. Discussion

The Michaelis-Menten is a kinetic model for enzymes, and the kcat/Km ratio is precisely the measure of performance [35]. Although the values of the Michaelis-Menten constant Km provide an approximation of the enzyme’s affinity for the substrate, and the kcat the number of molecules the conversion rate, it is the kcat/Km ratio that is the parameter that provides the overall catalytic efficiency [36]. RsAK has the best catalytic efficiency for the invertebrate organisms found, with a kcat/KmArg of 523 and a kcat/KmATP of 458 (Table 1). Catalytic conversion constants are a significant parameter for enzymatic performance, suggesting the organism’s metabolic behavior.
On the other hand, although the specific activity is in the order of that reported for other arthropods, this is the first report of a phosphagen kinase by a hematophagous ectoparasite. Furthermore, when R. sanguineus reaches the host, it can remain feeding on Other reports for AKs come from the cattle tick (Boophilus microplus) [37] and the cat flea (Ctenocephalides felis) [38] using proteomic approaches.
Antigenic sites are essential since these enzymes are proteins that may elicit an immune response, such as allergens. To date, there are algorithms capable of predicting both linear and discontinuous epitopes. It should be mentioned that 90% of the epitopes are discontinuous [39]. Ayuso [40] studied the linear epitopes of L. vannamei AK. As the sequences between the different AK species are remarkably conserved, we focused on using the DiscoTope algorithm to search only conformational epitopes and to find species-specific epitopes for RsAK. This was succeeded by comparing AKs from L. vannamei [20], P. pythagoricus [41], Scylla paramamosain [23] and Limulus polyphemus [22], since these structures were the ones with more identity with RsAK. The species-specific epitopes found in this work could serve as biomarkers of tick exposure since Rhipicephalus sanguineus is the primary vector of Rickettsia rickettsii, the causative agent of Rocky Mountain Spotted Fever. The results of the DiscoTope survey suggest that four species-specific epitopes for RsAK are present at the residues 157–169, 240–241, 257–258, and 335–341 (Figure 5). None of these sites appear to be related to enzymatic catalysis.
A feature of AK is the substrate-induced conformational change that occurs during the reaction, which has been determined by X-ray crystallography as the TSAC [14]. We observed a significant shift in the C-terminal domain, where the apo form of the enzyme is more open than the substrate/product (Arg-ADP) closed conformation, having an RMSD of 1.24 Å (Figure 3). Minor changes are observed in the N-terminal domain, where an RMSD of 0.28 Å was found between both conformations. All the above is consistent with López-Zavala et al. [31], analyzing the crystal structures of shrimp AK in the apo, binary complex with arginine, and in the substrate/product (Arg-ADP) closed conformation. These observations indicate that when AK binds to its substrates and cofactors, its shape is compressed, allowing a strong bond with its substrates. It also confirms that the active site cavity closes during catalysis and that a more compact conformer occurs during the reaction.
In conclusion, AK is an efficient biocatalyst due to its high kcat/Km parameters expressed in the tick [24]. The recombinant RsAK can be easily expressed and purified to a 20 mg/lt culture yield, suitable for structural and biotechnological applications. Since the essentiality of AK for invertebrates has been demonstrated [42], strategies for obtaining inhibitors may help to address the resistance to pesticides [43]. Future work will be focused on the RsAK enzymatic inhibition that may perturb the bioenergetic balance in the parasite and may lead to novel chemical strategies to control this pest, a problem for animal and human health.

4. Materials and Methods

4.1. Cloning and Expression of Recombinant RsAK

The procedure to obtain the bacterial clone expressing RsAK is depicted in Figure 6. The R. sanguineus nucleotide sequence was obtained from the brown tick’s AK transcript (GenBank: HACP01014757.1) [24] and optimized for recombinant expression in E. coli. A synthetic gene (gBlock, Integrated DNA Technologies (IDT), Coralville, IO, USA) encoding the RsAK amino acid sequence was synthesized for overexpression in E. coli. The gBlock sequence was optimized for expression in bacteria. It included an N-terminal tag of 6 histidine residues and two restriction sites, Nde I at the initial methionine codon and the BamH I restriction after the stop codon. The gBlock was initially cloned into the CloneJET system (Thermo Scientific, Waltham, MA, USA) and later subcloned into a pET11a expression vector (Novagen, Merck, Darmstadt, Germany) using the after-mentioned restriction sites. Cloning was confirmed by Sanger DNA sequencing at the University of Arizona GATC facility (Tucson, AZ, USA). E. coli strain BL21 (DE3) Rosetta II was transformed with 80 ng of the pET11a/RsAK construct. Then, 10 mL of an overnight culture of transformed bacteria was used to inoculate a Fernbach flask with 1 L of LB medium and antibiotics (100 µg/mL of ampicillin and 68 µg/mL of chloramphenicol). The bacteria were grown at 37 °C with an agitation speed of 225 rpm until they reached an A600 of 0.6. Isopropyl thiogalactopyranoside (IPTG) was added as an inducer to a final concentration of 0.2 mM. Bacteria were cultured for 18 h at 25 °C and harvested by centrifugation at 4500× g for 15 min.
The pellet was lysed by sonication on an ice bath, using three volumes of lysis buffer (2 mM Tris HCl pH 7.5, 1 mM DTT, 0.5 mM PMSF, 5 mM benzamidine, 500 mM NaCl, 0.1 mg/mL lysozyme, and 0.5 mM EDTA). The lysis was performed at a Branson® sonifier (Emerson, St. Louis, MO, USA) with a 50% duty cycle and a central output at level 5. Sonication was carried out for 15 s three times with an incubation period in the ice of 30 s between sonications. The lysate was centrifuged at 4500× g, and the supernatant was clarified with a 0.45 µm filter.
The recombinant protein was purified by metal ion affinity chromatography (IMAC) using a 5 mL Cytvia® HisTrap FF GE® column (Marlborough, MA, USA). First, the resin was equilibrated with five volumes of buffer A (20 mM Tris HCl, 500 mM NaCl, pH 7.4). Next, the sample was loaded into the column, and the column was washed with ten volumes of 4% buffer B (20 mM Tris HCl, 500 mM NaCl, and 0.5 M imidazole, pH 7.4). Finally, the His-tagged protein was eluted with 20% of buffer B at a 5 mL/min flow.
A second purification step was carried out with size exclusion chromatography (SEC) using a Cytvia® Superdex 75 10/300 GL column (Marlborough, MA, USA). The protein collected in IMAC was dialyzed to change the buffer from IMAC to SEC buffer (50 mM Tris HCl, 150 mM NaCl, pH 7.0) and concentrated using a 30 kDa Amicon® (MilliporeSigma, Burlington, MA, USA) by centrifuging the sample at 6000 rpm until it reached a volume of 2 mL. The chromatography was run at a flow of 0.5 mL/min. Elution peaks were analyzed on the chromatogram to corroborate purity and determine its oligomeric state using a standard curve.

4.2. RsAK Kinetic Characterization

The enzymatic activity of the RsAK was measured according to the methodology proposed by Blethen and Kaplan (1968) [26]. This assay consists of a coupled system in which the oxidation of NADH was measured spectrophotometrically at 340 nm. First, AK phosphorylates arginine and produces ADP, which is again phosphorylated by pyruvate kinase, producing pyruvate, which finally oxidizes NADH with lactate dehydrogenase. The reaction was carried out at 30 °C in a 0.5 mL quartz cell containing a final concentration of 178 mM glycine, 0.33 mM 2-mercaptoethanol, 133 mM potassium chloride, 13 mM magnesium sulfate, 20 mM phosphoenolpyruvate, 6.7 mM ATP, 0.13 mM nicotinamide dinucleotide (NADH), 2 U pyruvate kinase, 3 U lactate dehydrogenase, and 15 µg of RsAK equivalent to a final concentration of 0.37 µM in the reaction cuvette. The final concentrations of L-arginine substrate varied from 0–7.5 mM to determine the kinetic constants.
Blank with all reagents except RsAK enzyme was run. Absorbance data were collected for 5 min. Experimental data was collected in triplicates. One unit of arginine kinase activity is defined as the amount of enzyme that converts one µmol of L-arginine and ATP to N-phospho-L-arginine and ADP per minute at pH 8.6 and 30 °C, using the molar extinction coefficient of 6.22 mM−1 cm−1 for β-NADH at 340 nm. The final concentrations of the L-arginine substrate varied from 0–7.5 mM, and the concentrations of the ATP substrate ranged from 0.05–6.7 mM to determine the kinetic constants.
The kinetic parameters Vmax and Km for L-arginine and ATP were determined by fitting the initial reaction rates to the Michaelis-Menten equation model using non-linear regression analysis in GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).

4.3. Crystallization of apo RsAK

The protein was dialyzed in a 50 mM Tris HCl pH 7.8 buffer and concentrated to 11.5 mg/mL with an Amicon® membrane of 500 µL capacity, 10 kDa cut-off point at a speed of 12,000× g. Crystallization experiments were performed using the micro-batch technique in 72 well Greiner plates. The Hampton Research® kits (Aliso Viejo, CA, USA): Crystal Screen and Index kits were used to set up the micro-batch crystallization experiments. First, 1 µL of RsAK solution at 11.5 mg/mL was placed in each well, followed by 1 µL of the crystallization solution and 10 µL of paraffin oil. The plates were sealed and kept at 16 °C. Crystal formation was monitored under a stereoscope every week. After one week, RsAK crystals were obtained using the Index kit condition #43 (0.1 M BIS-Tris pH 6.5, 25% w/v polyethylene glycol 3350.) Next, an acupuncture needle was used to separate crystals and transfer them to a cryoprotectant solution (reagent #43 with 20% glycerol) for 5 min. Then the crystals were loop-mounted and flash-cooled at 100 K with liquid nitrogen for data collection.

4.4. X-ray Diffraction Data Collection and Crystal Structure Determination

Using an Eiger detector, data collection from RsAK crystals was performed on beamline 14-1 of the Stanford Synchrotron Radiation Light source (SSRL) at 1.19 Å wavelength. Crystal to detector distance was 150 mm, and the data collection was performed in a continuous rotation mode and consisted of 360 frames covering a total rotation range of 180 degrees. The frame images were integrated using the XDS program (Version March 15, 2019 BUILT=20190315) [44], the conversion to .mtz format was completed with COMBAT, and scaling was performed with SCALA, all from the CCP4 crystallographic software suite (Update 7.0.066) [45]. The RsAK crystal belongs to the P1 21 1 space group with unit cell dimensions of a = 48.41 Å, b = 61.41 Å, c = 61.34 Å, and β = 96.56°. Cell content analysis indicates one RsAK molecule per asymmetric unit with a Vm = 2.16 Å3 D−1 and a solvent content of 43% [46]. Phases were determined by molecular replacement with Phaser (release 2.7.17) [47] using L. vannamei AK structure (PDB 4AM1) as the search model. The amino acid identity between shrimp and the tick RsAK sequences was 79%. Several refinement cycles were performed with PHENIX (version 1.18.2-3874) [48], and COOT (release 0.9.4.1) [49,50] was used for manual refinement to obtain the final structure. The final experimental model and structure factors were deposited at the PDB under deposition 7RE6.

4.5. Molecular Modeling

The substrate/product (Arg-ADP) in a closed conformation was obtained by homologous modeling using the structure of shrimp L. vannamei TSAC (PDB 4BG4) [24] as a template using the Phyre2 algorithm [51].

4.6. Prediction of Species-Specific Discontinuous Epitopes with DiscoTope

To find species-specific epitopes of RsAK, the AKs deposited in PDB that present a higher percentage of identity with RsAK (L. vannamei, P. pythagoricus, and Limulus polyphemus), as well as that of Rhipicephalus sanguineus, were analyzed with the DiscoTope 2.0 algorithm [25] with a score threshold of −3.7. Predicted epitopes were contrasted with those found in Scylla paramamosain [18]. The AK structures of these species were aligned in PYMOL (version 1.8). The predicted epitopes for each species were mapped in their three-dimensional structure to see which amino acids conserve their antigenicity. The antigenic residues unique to Rhipicephalus sanguineus were marked as putative species-specific epitopes. Also, a multiple sequence alignment (https://tcoffee.crg.eu/ (accessed on 22 August 2022)) with the RsAK sequences, L. vannamei, P. pythagoricus, and Limulus polyphemus was conducted to map the epitopes of each species within the sequences.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/catal12101178/s1, Figure S1. Immobilized metal affinity chromatography (IMAC): Inset contains the SDS-PAGE electrophoresis analysis of selected fractions: Mwm: Molecular weight marker, Lane 1: Crude extract from E. coli, Lanes 2–9: fractions corresponding to the second peak. Figure S2. Size molecular exclusion chromatography (SEC). EV: elution volume in mL. Inset contains the SDS-PAGE electrophoresis analysis of selected fractions: Mwm: Molecular weight marker, Lane 1: Aliquot from the second peak from IMAC, Lanes 2–6: fractions corresponding to the main peak eluted at 11.64 mL.

Author Contributions

Conceptualization, A.C.G.-Y., M.A.I.-O., J.G.V. and R.R.S.-M.; methodology, A.C.G.-Y., E.N.M.-C., K.D.G.-O., A.L., M.A.I.-O., A.A.L.-Z. and R.R.S.-M.; software, A.C.G.-Y., K.D.G.-O., A.A.L.-Z. and R.R.S.-M.; validation, A.C.G.-Y., A.A.L.-Z. and R.R.S.-M.; original draft preparation, A.C.G.-Y. and R.R.S.-M.; writing—review and editing, all authors; visualization, A.C.G.-Y. and E.N.M.-C.; project administration, R.R.S.-M.; funding acquisition, R.R.S.-M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Consejo Nacional de Ciencia y Tecnología (CONACYT, México), grant number CF-2019-610264. CONACYT also provided a Ph.D. Scholarship to A.C. Gómez-Yanes and an internship scholarship (Ayudante SNI3) to E.N. Moreno-Córdova). A.L. acknowledges support from Argentina’s Agencia Nacional de Promoción Científica y Tecnológica (PICT-2017-0684). The Stanford Synchrotron Radiation Light Source, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). This publication’s contents are solely the authors’ responsibility and do not necessarily represent the official views of NIGMS or NIH.

Data Availability Statement

The crystal structure and structural factors for the RsAK are available at the PDB at the website: https://www.rcsb.org/structure/7RE6 (accessed on 22 August 2022).

Acknowledgments

We thank CTAOA-CIAD for sustained support and infrastructure.

Conflicts of Interest

The authors declare no conflict of interest.

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Catalysts 12 01178 g001 550
Figure 1. Michaelis-Menten plots of RsAK kinetics with L-arginine and ATP substrate. (A) represents the kinetics with L-arginine. The final concentrations of L-arginine varied from 0.078 mM to 7.5 mM. (B) represents the kinetics with ATP. The final concentration of ATP ranged from 0.05 mM to 6.5 mM. Data is described as means ± standard deviation of experimental triplicates.
Figure 1. Michaelis-Menten plots of RsAK kinetics with L-arginine and ATP substrate. (A) represents the kinetics with L-arginine. The final concentrations of L-arginine varied from 0.078 mM to 7.5 mM. (B) represents the kinetics with ATP. The final concentration of ATP ranged from 0.05 mM to 6.5 mM. Data is described as means ± standard deviation of experimental triplicates.
Catalysts 12 01178 g001
Catalysts 12 01178 g002 550
Figure 2. Crystallographic structure of the apo RsAK. (A) cartoon representation of RsAK with the N-terminal domain in green and the C-terminal in grey. (B) representative electron density of the 2Fo-Fc map at 1 σ. (C) representation of the arginine binding site. Interacting residues are shown as sticks.
Figure 2. Crystallographic structure of the apo RsAK. (A) cartoon representation of RsAK with the N-terminal domain in green and the C-terminal in grey. (B) representative electron density of the 2Fo-Fc map at 1 σ. (C) representation of the arginine binding site. Interacting residues are shown as sticks.
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Catalysts 12 01178 g003 550
Figure 3. Structural overlapping of the experimental structure RsAK apo (in red) with the theoretical RsAK substrate/product (Arg-ADP) closed conformation model (in green), showing ADP and Arg substrates as sticks.
Figure 3. Structural overlapping of the experimental structure RsAK apo (in red) with the theoretical RsAK substrate/product (Arg-ADP) closed conformation model (in green), showing ADP and Arg substrates as sticks.
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Catalysts 12 01178 g004 550
Figure 4. Identification of epitopes in the sequence and structure of AKs. (A): Epitopes predicted by DiscoTope shown in the structural models of invertebrates AKs (B): linear amino acid sequence alignment (panel B). Rs stands for R. sanguineus, Lv for L. vannamei, Pp for P. pytagoricus, Lp for L. polyphemus, and Sp for S. paramamosain. Colors match both structural models and linear amino acid sequences.
Figure 4. Identification of epitopes in the sequence and structure of AKs. (A): Epitopes predicted by DiscoTope shown in the structural models of invertebrates AKs (B): linear amino acid sequence alignment (panel B). Rs stands for R. sanguineus, Lv for L. vannamei, Pp for P. pytagoricus, Lp for L. polyphemus, and Sp for S. paramamosain. Colors match both structural models and linear amino acid sequences.
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Figure 5. Species-specific epitopes for RsAK.
Figure 5. Species-specific epitopes for RsAK.
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Figure 6. Scheme of the cloning and expression methods to produce RsAK.
Figure 6. Scheme of the cloning and expression methods to produce RsAK.
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Table
Table 1. Comparison of kinetic constants of invertebrate AKs.
Table 1. Comparison of kinetic constants of invertebrate AKs.
SpeciesRef.KmArg
(mM)		KmATP
(mM)		kcat
(s−1)		kcat/KmArg
(s−1mM−1)		kcat/KmATP
(s−1mM−1)		Vmax
(μmol min−1)
Rhipicephalus
sanguineus
(dog tick)		Present study0.27 ± 0.080.31 ± 0.12142 ± 0.21523.2458.126.1
Ascaris suum
(nematode)		[28]0.13 ± 0.0170.65 ± 0.05645.9353
Locusta migratoria
manilensis
(grasshopper)		[29]0.95 ± 0.081.29 ± 0.23159.4 ± 6.2169123.5
Polybetes pytagoricus
(spider)		[22]1.7 7544.4 27.8
Aphonopelma
(Dugesiella) hentzi
(spider)		[26]0.27
Cissites cephalotes
(beetle)		[30]1.01 ± 0.070.95 ± 0.162 ± 0.0522.13
Periplaneta americana
(cockroach)		[17]0.490.141.32.79.321.7
Table
Table 2. X-ray data collection and refinement statistics.
Table 2. X-ray data collection and refinement statistics.
ParametersRsAK
Data Collection Statistics
Space GroupP1211
Unit cell dimensions
a, b, c (Å)48.41, 61.41, 61.34
α, β, γ (°)90, 96.56, 90
Resolution range (Å)40–1.53
No. of reflections182,917 (24,964)
No. of unique reflections52,890 (4999)
Completeness (%)98.7 (95.3)
Rmeas (%)5.9 (37.2)
CC1/2 (%)99.9 (90.7)
I/σ (I)12.04 (2.87)
Multiplicity3.4 (4.9)
Monomers per asymmetric unit1
Refinement statistics
Resolution range (Å)40–1.53
Rwork/Rfree (%)15.52/18.57
Number of reflections52,782 (4966)
Clash score
Mean B-values (Å2)
Protein
Solvent
18.3
49.1
RMSD from ideal stereochemistry
Bond lengths (Å)0.49
Bond angles (degrees)0.69
Ramachandran plot (%)
Most favored region95
Additional allowed regions4.7
Disallowed regions0.3
PDB code7RE6
Values in parentheses are for the high-resolution shell (1.87–1.53 Å).
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Gomez-Yanes, A.C.; Moreno-Cordova, E.N.; Garcia-Orozco, K.D.; Laino, A.; Islas-Osuna, M.A.; Lopez-Zavala, A.A.; Valenzuela, J.G.; Sotelo-Mundo, R.R. The Arginine Kinase from the Tick Rhipicephalus sanguineus Is an Efficient Biocatalyst. Catalysts 2022, 12, 1178. https://0-doi-org.brum.beds.ac.uk/10.3390/catal12101178

AMA Style

Gomez-Yanes AC, Moreno-Cordova EN, Garcia-Orozco KD, Laino A, Islas-Osuna MA, Lopez-Zavala AA, Valenzuela JG, Sotelo-Mundo RR. The Arginine Kinase from the Tick Rhipicephalus sanguineus Is an Efficient Biocatalyst. Catalysts. 2022; 12(10):1178. https://0-doi-org.brum.beds.ac.uk/10.3390/catal12101178

Chicago/Turabian Style

Gomez-Yanes, Ana C., Elena N. Moreno-Cordova, Karina D. Garcia-Orozco, Aldana Laino, Maria A. Islas-Osuna, Alonso A. Lopez-Zavala, Jesus G. Valenzuela, and Rogerio R. Sotelo-Mundo. 2022. "The Arginine Kinase from the Tick Rhipicephalus sanguineus Is an Efficient Biocatalyst" Catalysts 12, no. 10: 1178. https://0-doi-org.brum.beds.ac.uk/10.3390/catal12101178

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