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FIELD OF THE INVENTION;

This invention relates to a process for engineering nucleotide specificity.

This invention further relates to a process for engineering nucleotide specificity of enzymes in material parasites by altering the electrostatic properties of gatekeeper residues outside the active site required for nucleotide specificity, without changing the active site residues of enzymes.

BACKGROUND OF THE INVENTION;

Enzyme engineering is a continuously evolving field and beneficial for desired applications in biochemistry like modifying substrate specificity, cofactor switching, changing affinity etc. Tailor made enzymes are the need of the for drug design, biochemical product synthesis on industrial scale. This study suggests that the functionally crucial features of the nucleotide selectivity advances rational inhibitor design against clinically relevant kinases, transferases/ synthetases. Nucleotide competitive inhibitor or nucleotide competitive kinase inhibitors can be designed based on nucleotide selectivity, Altering substrate specificity is one of the important properties used in protein engineering of enzymes. In the field of enzyme engineering, gatekeeper residues can be used to modify the different range of enzymes (synthetases, kinases, transferases) to show broad substrate specificity which is beneficial for industry and drug discovery. Novel enzymes can be engineered by completely switching the specificity as well as affinity towards the desired substrates. Studying the mechanism of nucleotide selectivity, ATP or GTP competitive inhibitor can be developed and may overcome the drug resistance problem since gatekeeper residues are away from active site of enzyme.

Understanding the role of gatekeeper residues acquired by sequence and structural information can be of profound importance, as this information may be used to engineer their role as desired. This contribution to our understanding of nucleotide selectivity of this enzyme serves as an important step for the structure guided design of competitive inhibitors derived from the mechanism of nucleotide specificity.

Significance in malana; SCS is an important enzyme, since it generates ATP through substrate-level phosphorylation in most of the organisms. Specifically in malaria parasite, SCS is required for de novo heme synthesis. The parasite synthesized heme is absolutely essential for parasite growth during the mosquito and liver stages. It seems SCS play crucial role in the life cycle of Plasmodium, and their gatekeeper residues may be targeted in drug W

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discovery for malaria, since P. falciparum SCS has different gatekeeper residues than human SCS.

There are land mark models like "lock and key ¹ " and "induced fit model ² " have revolutionized the field of enzyrnology. There has been lot of progress in the field of enzyme engineering which further suggests that this area of research is continuously evolving ³ " ⁶ . In a wider prospective molecular association of proteins with other proteins, DNA and ligands is thought to be driven by interaction free energies arising from structural features like hydrogen bonding and amino acid propensity ⁷ . A precise understanding of these interactions requires in-depth analysis of the factors governing these associations. Structural analysis of macromolecules and their interacting partners remains the most promising method lo decipher the rules governing these associations. Molecular recognition of cognate and non-cognate ligands by proteins is a well-known occurrence, normally explained by electrostatic and spatial complementarity but more complicating situation where proteins have to recognize between very similar ligands like Adenine (A) and Guanine (G) are still poorly understood. To explain the molecular recognition of A and G by nucleotide binding proteins, it was proposed that the distribution of hydrogen bond donors and acceptors in protein atoms as well as in complimentary purine rings might be used to differentiate ATP-specific binding sites from GTP-specific binding sites ⁷ . The insufficiency of this proposed mechanism to explain molecular discrimination led another group to re-examine the question of discrimination between A and G by nucleotide binding proteins. Focusing on electrostatic potential (ESP) of purine binding sites showed a clear correlation in ESP patterns and A/G specificity across proteins families ⁸ . This study established the role of a strong electrostatic component for molecular discrimination by calculating ESP of each binding substrate. Earlier discoveries also showed that individual amino acids do contribute in the overall electrostatic field of a protein which can be calculated by continuum solvent model ¹⁰ . The electrostatic properties of amino acids in the active site would be of considerable importance as changing the charge of the constituent amino acids in the catalytic site resulted in altered function and overall stability of the protein ¹⁰ . ¹¹ . But monitoring the effects of changing electrostatic properties of amino acids in the protein-ligand interactions at sites near or outside the active site still need prompt investigation as this is still a grey area of research in the field of enzyrnology. SCS is an important enzyme of TCA cycle. The substrate level phosphorylation reaction catalyzed by SCS, and takes place in three steps as described below;

E + succinyl-COA +Pi <→ E - succinyl-P03 + CoA (1)

E - succinyl-P03 <→ succinate +E-P03 (2)

E-P03+NDP <→ NTP+E(3)

The equilibrium constant of above reaction is approximately 1 , the direction of the reaction catalyzed by SCS depends on the local concentrations of nucleotides (NTD). SCS which is specific for ATP, involved in the citric acid cycle, will quickly respond to the energy changes of the cell since the ratio of ATP to ADP varies about 1 depending on the metabolic state of the cell. A GTP- specific enzyme is expected to maintain the high level of succinyl-CoA required by CoA transferase during ketone body metabolism since the value of the GTP/GDP ratio in mammalian mitochondria is around 100 or higher ²⁶ .

This idea is supported since a correlation has been shown to exist between the SCS activity that is specific for GTP and the utilization of ketone bodies ²⁶ .

In previous computational analysis using different models the interactions between succinyl-CoA- synthetase (SCS) and its ligands A and G ¹² has been studied. The analysis of SCS from Blastocysts, E. coli, and pig, provided important insights in understanding the role of amino acid residues outside the active site (Gatekeeper Residues). On the basis of information inferred from the above mentioned analysis, an "Electrostatic Gatekeeper mechanism" has been proposed without any experimental proof.

SCS can be considered a serendipitous example in this respect as there are forms of the enzyme with known co fact or specificities for ATP, GTP, or both. Referring to its nucleotide specificity, SCS enzyme has different isoforms in different species as prokaryotic SCS is capable of using both nucleotides ATP/ GTP, but in eukaryotes SCS has ATP specific or GTP specific isoforms ^{13 18} .

SCS is composed of two subunits SCSa and SCSβ, but only the β subunit has nucleotide binding site. SCS appear as dimers in eukaryotes while prokaryotes have hetrotetrameric form ^{14 17} . Previous reports indicate that in Blastocysts, SCS recycles succinyl-CoA by acetate-succinate CoA transferase ¹⁹ . In addition, SCS acts as an entry point for valine and isoleucine catabolism. Since, hydrogenosomes do not have the ability to generate energy though oxidative phosphorylation, SCS is particularly important in these organisms, as it generates ATP through substrate-level phosphorylation ¹² .

SCS has also been shown to generate succinyl-CoA for the first reaction of heme biosynthesis in mosquito and liver stages of malaria parasite ²⁰ . In malaria parasite, both asexual and sexual blood stages have a conventional TCA cycle to catabolize glucose as well as glutamine. In sexual stage, gametocyte differentiation is associated with a programmed remodeling of central carbon metabolism that may be required for parasite survival either before or after uptake by the mosquito vector ²¹ . P. falciparum asexual and gametocyte stages catabolize host glucose and glutamine in mitochondria via TCA cycle. The main carbon fluxes around the TCA cycle in the asexual stages are driven by input of carbon skeletons urinating from glutamine and SCS play important role to control the fluxes of glutamine and malate ²¹ .

Unlike SCS, aminoglycoside 2 phosphotransferase IVA controlled nucleotide selection by residues at the active Site ²² . Similarly, in aminoglycoside 2-Phosphotransferase-IIa, bulky tyrosine at the active site blocks the access of ATP ²³ , but in case of SCS, only charge residues away from the active site control the nucleotide selectivity. In case of DNA polymerase μ, unlike SCS, active site gatekeeper residues help to discriminate non-cognate nucleotides ²⁴ . Further, in case of tyrosine kinases, inactive enzyme can be activated by mutating a gatekeeper residue (threonine) at the active site and hydrophobic spine can be created by enzyme engineering which cause kinase inactivation ²⁵ .

Being a TCA cycle enzyme, SCS has been studied in detail, its biochemical characterization, and crystal structures have been determined from various organisms ¹⁴ " ¹⁸ , but the factors governing nucleotide specificity have not been explored in detail.

OBJECTS OF THE INVENTION;

It is therefore an object of this invention to propose a process for engineering nucleotide specificity.

It is a further object of this invention to propose a process for engineering nucleotide specificity, for providing an additional level of substrate specificity of enzymes. Another object of this invention is to propose a process for engineering nucleotide specificity, which is simple and cost-effective.

These and other objects of the invention will be apparent to a reader on reading the ensuing description, in conjunction with the accompanying drawings.

SUMMARY OF THE INVENTION:

According to this present invention, there is provided a method for engineering nucleotide specificity comprises: i) an amplification of succinyl- CoA (SCS) in two forms named SCSa and SCS from CDNA and cloned into expression vector; ii) clones containing two forms of SCS were sequenced to check orientation by transforming positive clones of SCSa and SCS into E.Coli codon plus component cells with PET 28 a vector; iii) 2 liters of LB media containing 50 g/mL kanamycin and 34μg/mL chloramphenicol was inoculated with glycerol stocks to SCS grown at 37°C to O.D at 600 0.6 to 0.8; iv) ImM of Isopropyl β-D-l-thiogalactopyranoside (IPTG) was added to protein expression and cultures were frown for 3:30 hrs after induction at 37°C and subjected to centrifugation for a substantial period such as herein described; v) collecting of supernatant after centrifugation and further subjected to purification and refolding mechanisms;

- said SCS proteins were extracted from Blastocystis and P. falciparum;

- said primers are used for cloning of SCSa are herein given below: a) Blastocystis SCSa

i) FP: 5' -agaaga GGA TCCGAGCAG CACTGCCCGTGTGTGGG-3' ii) RP: 5 ^f -tcttctGCGGCCGCTTAAGCCTTT CCAGCAGCCTTC-3' b) Blastocystis SCSfi

i) FP: 5'-agaagaCA ^4TOCTGAGAATGGCCCCTAAGACTGTG-3' ii) RP: 5'-tcttctGTCGACGTGCTTCAGAGAAGCAACAGCC-3' c) Δ P. falciparum SCS (KY) (Gatekeeper mutant)

i) FP: 5'-TGAACGTTTTAAGATAAGAAAAGAAAGATATATT-3' ii) RP: 5'-CAAAGTTTTGTAATAATAAAGC-3' d) Δ P. falciparum SCS (DE) (Gatekeeper mutant)

i) FP: 5'-TGAACGTTTTGAGATAAGAAAAGAAAGATATATT-3' ii) RP: 5' -TTACTAACTTATTATCACC-3' e) Δ P. falciparum SCS (KK) (Gatekeeper mutant)

i) FP: 5'-TGAACGTTTTAAGATAAGAAAAGAAAGATATATT-3' ii) RP: 5'-TTACTAACTTATTATCACC-3'

- said centrifugation has occurred at 3500g for 20min at 4°C.

- said refolding process comprises of mixing of SCSa and SCSp with equal proportion and diluted by 100 fold with buffer containing 50mM Tris-cl at pH8, 25% glycerol, 25mMDTT, ΙΟΟμΜ MgCb, 50mM ATP.

- said refolded protein broth incubated overnight at 4°C with mild stirring;

- said refolded protein broth was centrifuged at 15000rpm for 30min and thereby concentrated using 10 kD cuff centricon;

- said final cone, in folding buffer was 50μg/mL. BRIEF DESCRIPTION OF THE INVENTION

According to this invention is provided a process for engineering nucleotide specificity.

In accordance with this invention, two forms of succinyl-CoA synthetase (SCS) from two different parasites (Blastocystis and P. falciparum), have been used to engineer the substrate specificity by altering the electrostatic properties of the gatekeeper residues outside the active site. The gatekeeper resides in SCS were identified and using site directed mutagenesis and enzyme kinetics the inventors have demonstrated that gatekeeper residues outside the active site are required for nucleotide specificity. In Blastocysts SCS, the mutation in gatekeeper residues (KEKD) resulted ATP-specific-SCS can now bind to GTP with high affinity. Moreover, mutation near the active site (VLLF) had no effect on GTP activity but with the gatekeeper residues still acting to preclude binding. However, combining a gatekeeper mutant with near active site mutant (KEKD+VLLF), a complete reversal of nucleotide selectivity was obtained with a GTP affinity but no detectable activity with ATP. Further, P. falciparum SCS was used as another model since it is different than Blastocystis SCS, and "Gatekeeper effect" has been reinvestigated. In summary, using molecular modeling, deciphering electrostatic properties of the surface residues, molecular dynamics stimulation and finally using mutagenesis and enzyme kinetics, the substrate specificity has been engineered by altering the electrostatic properties of the gatekeeper residues outside the active site.

Methodology of the invention

All known structures of representative SCS proteins (2FP4, 1CQI, 1EUD, 3UXF and 1SCU) were retrieved from the Protein Data Bank (www.rcsb.org) and aligned using the structure alignment program STAMP ²⁷ - ³⁰ . A profile hidden Markov model of the resulting alignment produced using the program hmmbuild from the HMMER 3.1 ³¹ suite was used to map the sequence of beta succinyl-CoA synthetase of Blastocystis and P.falciparum. The region corresponding to residues 15-416 and 50-447 of SCS β subunits of Blasiocystis and P. falciparum, respectively aligned to the profile. Two model structures for both the enzymes (β SCS of Blasiocystis and P. falciparum) one with GTP and other with ATP as ligand, were generated by Modeller 9vl032 using pig PSCS structure as template. To build an initial model of GTP- P.falciparum SCS subunits coordinates for GTP molecule was introduced into the model from the template (2FP4) and to build an ATP-P. falciparum SCSp subunits model, ATP molecule was constructed from ADP in the ADP-bound form of E.coli SCS (1CQI). Electrostatic surfaces were produced using the EF-surf server and visualize in PDBj viewer ³³ .

In order to observe protein-ligand interactions more realistically, the inventors carried out simulations for ATP & GTP starting from the predicted complex models. Mutant complexes (K58E/K127D) and (K58E/K127D+VI26L/L240F) were generated using Pymol ³⁴ for both ATP and GTP. The parameters for ATP and GTP were taken from AMBER ³⁵

Parameter Database;

http://www.pharmacy.manchester.ac.uk/bryce/amber/) followed by conversion to GROMACS ³⁶ format using Acpype ³⁷ . For ATP (wild/mutant/combined mutant) and GTP (wild /mutant/ combined mutant) complex formation, a preliminary energy minimization step with a tolerance 500 kj/mol/nm was run with the Steepest Descent method. All bonds were constrained using P-LINCS. After minimization, a short NVT (fixed volume and temperature) MD simulation (200 psec) with position restraints applied to each system to soak the macromolecule into the solvent. A time step of 2 fs was used in all cases, and the systems are coupled to a temperature bath at room temperature using V-resclae, a thermostat that uses velocity rescaling with a stochastic term. Long-range electrostatics was handled using the PME method. Cut-off was set at 1.0 nm for Coulomb interactions, and at 1.0 nm for Vander Waals interactions. This short MD was followed by another (Ins) MD in NPT conditions (fixed temperature and pressure), scaling down the position restraints in order to equilibrate the system. A pressure of 1 bar was coupled using the Berendsen's method. Finally, a 10 ns MD Simulation was performed separately for each system, time- step of 2.0 fs and without any position restraints. All other conditions were kept identical as the short NPT dynamics. Trajectories and energy components all were written at every 100 ps.

The specificity of a molecule is determined by calculating the Binding Affinity of ATP and GTP for both wild and mutant complexes. g_mmpbsa, a programme developed in our lab was used to calculate bio molecular associations like protein-protein, protein-ligand protein-DNA etc using MM- PBSA. It gives net Binding energy (Electrostatic +Evdw +Epol +Eapol).

(http://rashmikumari.github.io/g_mmpbsa/Usage.html#g mmpbsa) ³⁸ .

Cloning, expression and purification of SCS

SCS consists of two subunits SCSa and SCSβ. SCSa and SCS genes were separately amplified from cDNA and cloned into expression vector pET 28a (Novagen). A List of different mutations in SCS and their mutagenic primers is shown in Table 1. All mutants were generated by using site directed mutagenesis kit (New England Biolabs). Both SCSa and SCS subunits were cloned into pET 28a vector separately using appropriate restriction enzymes as mentioned in table 1 (restriction sites were mentioned as underlines) Table 1; list of primers used for cloning of SCSa and SCSp subunits from Blastocystis and P. falciparum.

A list of gatekeeper residues and their resultant net charge at the surface of active site of enzymes are shown in table 2.

Table 2; List of gatekeeper residues and their resultant mutant with net charge at the surface of active site in Blastocystis

Enzyme Wild type residue Mutated residue Result of mutation

Gatekeeper mutant Lysss and Lys ¹²⁷ Glu ⁵⁸ and Asp ¹²⁷ Negative charge (KEKD)

Near active site Vali2 ⁶ and Leu ²⁴ ° Leu ¹²⁶ and Phe ²⁴⁰ Similar to Pig SCS mutant (VLLF)

Combined mutant Lys ⁵⁸ and Lys ¹²⁷ Glu ⁵⁸ and Aspi ²⁷ Negative charge and (KEKD + VLLF) Val ¹²⁶ and Leu ²⁴⁰ Le ¹²⁶ and Phe ²⁴⁰ Similar to Pig Table 3: Gatekeeper mutants designed in case of P. falciparum SCS

All clones containing SCSa wild SCSp and different mutants were sequenced to check orientation and ORF for protein expression. Positive clones of SCSa wild SCSP and different mutant of Blastocystis and P. falciparum were transformed in E. coli codon plus (Novagen) component cells with pET 28 a vector. LB media (2 liters) containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol (Amresco) was inoculated with glycerol stocks of wild and mutants of SCS grown at 37°C to O.D at 600 0.6 to 0.8. Protein expression was induced by adding IPTG (ImM) and cultures were frown for 3.30 hrs after induction at 37°C. Cells were pelleted by centrifugation at 3500g for 20 min. at 4°C and resuspended in lysis buffer (50 mM phosphate buffer pH 8.0, 0.3 M NaCl). Cell bursting was done repeated freeze-thaw cycles and Ultrasonic processor (Biochem Life Sciences). Supernatant was collected by centrifugation at 3500 g for 20 min. at 4°C. The SCSa subunits of Blastocystis and P. falciparum were expressed as soluble protein, and further purified by Ni-NTA affinity chromatography as done earlier ^{36 39} . Pellet was processed for isolation of inclusion bodies (IBs), because SCS subunits of Blastocystis and P. falciparum were expressed as IBs. The purification of SCS subunits were purified as described as earlier ³⁶ " ³⁹ . After SDS-PAGE analysis purified fractions were pooled and concentrated by 10 kD cutoff Centricon (Vivaspin). Buffer exchange was done with 6M Gn-HCl, 50 mM Tris-Cl, pH 8.0 to a final volume of 1 ml.

Refolding of enzymes by rapid dilution method

Different refolding conditions were tried for purified denatured SCSa and SCS from both model systems (Blastocystis and P. falciparum), and different refolding conditions were used as described earlier ^{36 40} . The maximum activity of enzymes were obtained when SCSa and SCSβ subunits were finally mixed in equal ratio and refolded by 100 fold dilution in optimized refolding buffer containing 50 mM Tris-Cl, pH 8.0, 25% glycerol 25 mM DTT, 100 μΜ MgCl ₂₎ 50 μΜ ATP . Final protein concentration in folding buffer was 50μg/ml and incubated overnight at 4°C with mild stirring. Refolded protein was centrifuged at 15000 rpm for 30 min. at 4°C and concentrated using 10 kD cut off Centricon (Vivaspin).

Western blot analysis

Purified wild type and mutant enzymes were resolved by SDS-PAGE on 12 % acrylamide gels and transferred to nitrocellulose membrane (Whatman). For both the subunits the membrane was incubated with anti-His mouse monoclonal antibody (1:5000) followed by anti-mouse HRP conjugate (Sigma) (1:8000). The SCSa and SCS subunits were confirmed by anti-His monoclonal antibody conjugated with HRP. 3, 3-diaminobenzidine (Amresco) (lmg/ml) was used as a chromogenic substrate in western blot.

In our earlier patent report, we have submitted the initial rate of reactions for P. falciparum wild type and gatekeeper mutants. Now we are submitting the kinetics analysis of the wild type P. falciparum SCS and two gatekeeper mutants (KY and EY) (Table 3).

Kinetic analysis of wild type and Gatekeeper mutants of P. falciparum SCS

As reported in the previous patent, P. falciparum SCS has different gatekeeper residues than Blastocystis SCS. Gatekeeper residues of P. falciparum SCSp subunit are Asp64 and Tyrl64, which are negative and hydrophobic. In the earlier patent report, preliminary data showed that P. falciparum wild type SCS enzyme can utilize both the nucleotides at 100 mM cone. But our recent kinetics analysis consistently showed that P. falciparum SCS is ATP specific only, with a K _m =48.46 μΜ (Fig 1). Regarding the first gatekeeper mutant KY, there is moderate positive charge at the gatekeeper region due to Lys, and the K _m =61.32 μΜ (Fig. 1), corresponds to the concept that positively charged Lys supports ATP at the gatekeeper region. Our second gatekeeper mutant EY, however possess a negative amino acid (Glu) at the gatekeeper region with K _m - 84.106 μΜ (Fig 1), enzyme is still ATP specific only, but with a reduced affinity as compared to wild type and first gatekeeper mutant KY.

Discussion

According to our hypothesis, gatekeeper residues can be mutated to engineer nucleotide specificity without altering the binding site residues. We have designed single gatekeeper mutants of P. falciparum SCS to see the effect of change in charge at gatekeeper region as in the case of Blastocystis (mentioned in the patent report), in which positively charge at the gatekeeper region supported ATP and negative residues at the gatekeeper region supported GTP. The result of single gatekeeper mutant (KY), by changing negatively charged Asp to positively charged Lys, showed only a slight decrease in ATP affinity (K _m =61.32±l l μΜ) as compared to wild type (Κπι=4.46±10μΜ). In second gatekeeper mutant (EY), Asp has been changed to glu to mimic the gatekeeper residue in Pig. This mutant (EY) has a reduced affinity for ATP (Κπι=84.16±16μΜ) as compared to wild type and first gatekeeper mutant (KY). Though, these gatekeeper mutations are single and therefore a moderate effect on nucleotide specificity was evident. Kinetic analysis of P. falciparum wild type SCS and gatekeeper single mutants did not show the expected change as in case of Blastocystis, therefore we have further planned to see the gatekeeper effect with double mutant (both the residues change at the gatekeeper region). This change in gatekeeper residues should have more prominent effect on nucleotide specificity.

Enzyme Kinetics

Kinetics of wild type and mutant enzymes from both model systems were carried out as described earlier ¹² - ^{15 16} with some modifications. Composition of assay buffer was 129 μΜ of CoA, lOmM Sod. Succinate, 50mM KC1, lOmM MgCl ₂ , 50mM Tris-Cl, pH 7.4. All wild type and mutant enzymes were quantitated by Bradford assay. Recombinant wild type and mutant enzymes were analyzed for kinetic parameters with given concentrations of nucleotides. In each assay of Blastocystis enzymes, 3 micro molar concentration of enzyme was used. For P. falciparum, initial rate of reaction for native enzyme was assayed from parasite lysate having 5 ml 3D7 culture with 5-6 % parasitemia, for initial rate of recombinant enzymes, 4 micro gram of enzyme was used in each reaction. The reaction was specifically followed by formation of Succinyl-CoA bond at 232 nm. Km values were calculated from three independent experiments using the Graphpad prism 5.

Identification of gatekeeper residues by homology modeling of SCS based on solved structure

To investigate the molecular basis of nucleotide specificity in Blastocystis SCS and P. falciparum SCS, different homology models were constructed using X-ray structures of pig SCS$, E. coli SCSfi subunits as templates. The "Electrostatic Gatekeeper mechanism" has been validated by molecular modeling, induced fit docking, mutagenesis and enzyme kinetics. Previous published data suggested that pig SCS is GTP specific ¹⁶ _' ¹⁹ , Blastocystis SCS is ATP specific ¹² , and E.coli can interact with both the nucleotides ¹⁴ . ¹⁷ . IT Modeling study suggested that Pig SCS has two negatively charged residues at the gate of active site, and on the other hand Blastocystis SCS has two positively charged residues corresponding to Pig. Positively charged (Lys58 and Lysl27) residues are present in Blastocystis SCS while in pig the corresponding residues are negatively charged (Glu78 and Asp 147). In E.coli, Pro34 and Asp 134 at the active site entrance while P. falciparum contains Asp94 and Tyrl64 at corresponding positions as gatekeeper residues. In E.coli and P. falciparum, the corresponding residues are negative and hydrophobic.

"Electrostatic Gatekeeper'" hypothesis

Based on above observation, it is proposed that GTP is restricted from binding to an ATP-specific succinyl-CoA-synthetase by a mechanism called "Gatekeeper effect", where charged, solvent exposed residues at the entrance to the active site produce electrostatic dipole interactions with approaching substrates, and control their access. Model is showing ATP specific SCS with positively charged residues at rim of the active site favoring only Adenine. Further, GTP specific SCS with negatively charged residues at rim of the active site favoring Guanine. Based on this model it was predicted that electrostatic potential of the rim surrounding the active site is responsible for determining nucleotide selectivity. These charged residues are named as "gatekeeper residues".

Electrostatic energy change for ATP and GTP in Blastocystis SCS

In order to observe the enzyme-substrate interactions more realistically, the inventors have carried out unbinding simulations for ATP and GTP starting from the predicted complex models. MD simulations were carried out for the wild, gate-keeper and combined mutants with ATP and GTP as a substrate. The unbinding trajectories of the two nucleotides were plotted as energy vs. time scale graphs for the wild, gate-keeper and combined mutants. Lower energy represents the greater stability towards the enzyme-substrate complex. Binding energy estimation for all complexes reveals that wild-ATP has lower electrostatic and polar solvation energy than wild GTP (Table 4). In case of gatekeeper complexes with ATP and GTP, gatekeeper- GTP has lower binding energy than gatekeeper-ATP (Table 3). The last complex, a combined mutation-GTP has lower binding energy than its counterpart combined mutation-ATP (Table 3), where ATP is not favored with a positive binding energy whereas GTP has negative binding energy, stating GTP is bound to the enzyme. Table 4: Energy Values of different enzymes of Blastocystis

Production of wilds and mutants SCS as recombinant enzyme

To investigate the "Electrostatic Gatekeeper" hypothesis, wild and mutants SCS were designed. In case of Blastocystis, a gatekeeper mutant (KEKD or K ⁵⁸ E, K ¹²⁷ D) was made by replacing K ⁵⁸ and K ¹²⁷ into negative charge residues (E ⁵⁸ and D ¹²⁷ ). the inventors further mutated residues closer to the active site (VLLF or V ¹ 26L, L ²⁴⁰ F) by replacing V and L ²⁴⁰ into Leu ¹²⁶ and Phe ²⁴⁰ respectively, making these residues similar to pig SCS. A combined mutant (KEKD+VLLF or K ⁵⁸ E, K ^{1 7} D+V ¹²⁶ L, L ²⁴⁰ F) was also designed to see cumulative effect of charge of gatekeeper residues and mutation near binding site of nucleotides. For P.falciparum, wild (D Y) and all the mutants ( Y, DE, KK) were generated as described earlier (Table 4). Table 5: List of gatekeeper residues and their resultant mutant with net charge at the surface of active site in P. falciparum

All the mutants were made by site directed mutagenesis and confirmed by DNA sequencing. All the wild and mutants enzymes of Blastocystis and P. falciparum were produced as recombinant form using E.coli expression system. Blasiocystis and P. falciparum SCSa were expressed as soluble form in E. coli, SCSa His-tagged subunits were purified using Ni-NTA affinity chromatography, while his tagged SCSp subunits were expressed predominantly as IBs, and SCS subunits were first isolated as IB and further purified by Ni-NTA affinity chromatography. Purified SCSa and SCSβ subunits of wile and mutant enzymes were pooled and concentrated in denatured medium as discussed in material methods. Both the subunits were refolded together using optimized refolding buffer as discussed in material methods. Active enzymes were further co-purified as α-β complex by affinity chromatography before measuring final Km.

Enzyme Assays for wild type and mutants

Assay buffers and other experimental condition for kinetic measurements of wild type and all mutants of SCS were performed as described earlier ¹² . K _m of the wild Blastocystis SCS for ATP was 143μΜ, while it did not show any delectable activity with GTP, However, after altering the charge of gatekeeper residues, mutant enzyme (KEKD) showed a higher affinity for GTP (Km=135uM) compared to ATP (Κ _π1 -247μΜ). These results supported the hypothesis that negatively charged gatekeeper residues preferred GTP as in case of pig SCS. While near active site mutant VLLF showed a clear preference for ATP as of wild type but with a lower affinity with Km of 267μΜ. Kinetic of VLLF mutant suggested that gatekeeper mutant (KEKD) showed a specific affinity for GTP. The strongest effect was observed after changing the gatekeeper residues and altering the residues near binding pocket. The combined mutant (KEKD+VLLF) showed a complete reversal of specificity as the enzyme showed a high affinity for GTP, however, no major detectable activity was observed for ATP (up to 1 mM) and K _m value for GTP was 124μΜ. MD simulation results also support the results of enzyme kinetics.

SCS is a very important enzyme in malaria parasite as it has been shown to generate succinyl-CoA for the first reaction of heme biosynthesis in mosquito and liver stages of malaria parasite. It has been previously reported that de-novo heme biosynthesis is important for parasite survival in mosquito and liver stages and targeting SCS would be a promising approach for control of liver stage of malaria parasite in humans. SCS has been found to possess different gatekeeper residues in Human and P. falciparum for controlling nucleotide access as mentioned in our previous patent report. Blocking of nucleotide access by designing inhibitors based on interactions with gatekeeper residues shall be a good strategy for inhibiting SCS activity.

Since P. falciparum SCS has different kind of gatekeeper residues compare to pig and Blastocystis, the inventors further used this enzyme as another model system to validate our hypothesis. Surface of nucleotide binding region of P. falciparum is neutral like E.coli, therefore, the inventors made three different mutations to see the gatekeeper effect in P. falciparum SCS.

The native SCS from P. falciparum lysate was assayed for nucleotide specificity as described earlier ¹² . Initial rate of reactions were recorded for both nucleotides, and were comparable. The inventors also recorded the initial rates for E.coli SCS as a control. Similar to the data previous literature also suggested that both the nucleotides can interact with this enzyme but ATP has slightly better affinity compare to GTP. The native SCS assay of P. falciparum and E.coli suggested that SCS can interact with both the nucleotide. The comparison of initial rate of wild and P.falciparum SCS mutant (KY) indicated that there was no significance change of nucleotide activit. Interestingly, the last two mutants where negative (DE) and positive charges (KK) were dominant showed a significance difference in nucleotide binding pattern. These results clearly suggested that gatekeeper residues control the access of nucleotide binding similar to Blastocystis SCS.