JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION

https://doi.org/10.1080/10799893.2020.1835960

ORIGINAL ARTICLE

Understanding the structural insights of enzymatic conformations for

adenylosuccinate lyase receptor in malarial parasite Plasmodium falciparum
Esam S. Al-Malki
Department of Biology, College of Science in Zulfi, Majmaah University, Majmaah, Saudi Arabia

ABSTRACT ARTICLE HISTORY

The dreadful disease malaria is one among the infectious diseases that comes in third number after Received 15 August 2020
the tuberculosis and HIV. This disease is spread by female Anopheles mosquito and caused by the Revised 9 October 2020
malarial parasite sp notably Plasmodium falciparum. In this, the organism has several enzymes for proc- Accepted 9 October 2020
essing the infection and growth mechanism and among that, the adenylosuccinate lyase is an enzyme
KEYWORDS
that plays a critical role in metabolism and cellular replication via its action in the de novo purine bio- Enzyme kinetics;
synthetic pathway. Adenylosuccinate has been studied for two reaction mechanisms, and in that, the phylogenetic analysis;
adenylosuccinate to AMP and fumarate is core important. As of now, there have been several studies reaction mechanism;
indicating the reaction mechanism of adenylosuccinate lyase, this study projects the conformations of homology modeling;molec-
the reactant and product changes through molecular docking and molecular dynamic simulations. ular dynamics; Malaria
Adenylosuccinate bound complex involves His role in the product than the reactant complex, and the
complex shows high flexibility due to fumarate. Thus, identifying the core inhibitor that binds to His
rings could be a standard adenylosuccinate lyase inhibitor, that can block the malarial diseases in
humans. In addition to the competitive inhibition site, we also predicted the uncompetitive ligand
binding site, which suggest the alternate region to be targeted. Thus, from this work, we suggest
both competitive and uncompetitive binding regions for the purpose identifying the malar-
ial inhibitors.

Introduction febrile paroxysms typical of the disease [9]. The parasite

infected red blood cells tend to sequester in the deep vascu-
In the infections caused by vector-borne disease, malaria
lature and thus avoid being cleared from the host in the
stands top with over 3.4 billion people, and notably, malaria
spleen, resulting in the circulatory blocking, inflammatory
is the leading disease that causes the child death [1,2]. Even
responses that could result in organ failure or death [10].
though the mortality rate decreases due to improvement
Malarial parasites have several drug targets, and among
measures by controlling vectors and implementation of arte-
them Purine Biosynthetic Pathway is mainly targeted for its
misinin combination therapies (ACT) [3]. But, all of sudden
core importance in the replication and metabolism mechan-
the drug resistant malaria infections cross over the measures
taken and resistant to malarial drugs [4]. In humans, the mal- ism [11]. This pathway includes several enzymes, that
aria is commonly caused by parasitic protists of the genus includes adenine phosphoribosyl transferase, AMP deami-
Plasmodium, and specially the species called P. falciparum, P. nase, 30 ,50 -nucleotidase, adenosine deaminase, purine nucleo-
vivax, P. ovale, P. malariae, and P. knowlesi. Among these spe- side phosphorylase, hypoxanthine-guanine phosphoribosyl
cies P. falciparum is the virulent and common sp that causes transferase, adenylosuccinate synthase, adenylosuccinate
malaria in most of the places [4]. In this spread, the female lyase, IMP dehydrogenase and adenosine kinase [12]. In this,
anopheles mosquito serves as the carrier for transmitting the adenylosuccinate lyase gene sequences have shown distinct
malaria parasite into the host human cells [5]. The mosquito homolog with the human adenylosuccinate lyase sequence,
saliva holds special features to carry the sporozoites and and therefore it is considered as one of the drug targets to
while the mosquito bites the humans, the saliva transmits inhibit the malarial parasite [13,14]. The adenylosuccinate
the sporozoites inside the skin, and then migrates to the lyase has a dual reaction mechanism, and in that adenylosuc-
liver [6]. Based on the host immunity, the time dependence cinate to AMP and fumarate is essential for metabolic activity
of infecting the RBC’s and new parasite progeny generated [15]. As of now, there are several studies indicating the reac-
in 48 h to 72 h [7]. This also varies from species to species of tion mechanism of adenylosuccinate lyase enzyme, but the
plasmodium, and lethal species infect much faster than the structural aspect of the reaction mechanism is still lacking.
others [8]. Especially, P. falciparum are highly lethal in the So that, through this study the detailed mechanical insights
plasmodium family, and they emerge to induce the cyclical of adenylosuccinate lyase enzyme is detailly studies using

CONTACT Esam S. Al-Malki [email protected] Department of Biology, College of Science in Zulfi, Majmaah University, Majmaah 11952, Saudi Arabia
Supplemental data for this article can be accessed here.
! 2020 Informa UK Limited, trading as Taylor & Francis Group
2 E. S. AL-MALKI

the homology modeling, molecular docking, and molecular considered as a grid for molecular docking [31]. Finally, the
dynamics studies. ligand with reactant and product is allowed to dock using
the auto dock and the scoring methods are used to allow
the evaluation of conformation states [32].
Materials and methods
MSA and phylogenetic analysis
Molecular dynamics simulations
A P. falciparum protein sequence of adenylosuccinate lyase
The complex from the auto dock is visualized using the chi-
protein sequence (Q7KWJ4) is retrieved from uniprot and
mera and pdbsum for the interactions and the whole com-
their similar corresponding proteins sequences were identi-
plex is subject to molecular dynamics simulations using the
fied and downloaded from GenPept [16]. For multiple
Desmond molecular dynamics package [33,34]. Initially, for
sequence alignment (MSA) the template and similar adenylo-
environment adaptability, the whole complex is optimized
succinate lyase protein sequence from various organisms are
using protein preparation with OPLS-FF. The system is set to
downloaded and the multiple sequence alignment is per-
surround the reactant and product with a TIP3P water model
formed using the PRALINE tool (with the Blosum62 matrix)
and the distance between the box is set to 10 Å from each
[17,18]. Phylogenetic tree is constructed using the Clustal-
side. For neutralization, the NaCl is added with 0.15 concen-
omega [19]. Here the phylogenetic calculations are per-
tration and systems are built. The built systems are subject
formed with the backend algorithm of neighbor-joining
to system minimization for 100ps and the standard MD
method as described by Saitou and Nei and the tree is
protocol is followed for system equilibration [35]. The final
visualized using the interactive tree of life [20].
MD step is performed for the 30 ns of the timescale using
the OPLS-FF and the trajectories are extracted for the pur-
Homology modeling pose of RMSD values using the Desmond simulation inter-
action analysis and VMD.
The P. falciparum protein sequence of adenylosuccinate lyase
protein sequence (Q7KWJ4) is retrieved from uniprot and the
sequence is searched for its suitable experimental template Results and discussion
through the Basic Local Alignment Search Tool (BLAST) with
MSA and phylogenetic analysis
reference to protein data bank (PDB) [21]. As the experimen-
tal structure is lacking, the closest similarity with maximum The Multiple sequence alignment (MSA) of adenylosuccinate
identity, lower e-value, similar function, and closely related lyase from P. falciparum along with similar functional sequences
phylogeny template protein with PDB ID: 2QGA (adenylosuc- from the different organisms are aligned. The aligned sequence
cinate lyase protein from P. vivax) is chosen as template information shows the input sequence is aligned with
structure [22]. The template and target sequence are extremely similar patterns, having more conserved regions.
matched with sequence alignment using Clustal-omega and Functionally and sequence wise similar patterns in alignments
the alignment is taken for modeling the protein using the show these sequences make them good candidates for analyz-
modeler 9v10 software [23]. The modeled structure of adeny- ing the evolutionary studies. Based on the MSA, the phylogen-
losuccinate lyase is manually imported in PDBsum (http:// etic tree with target sequence and the functionally similar
www.ebi.ac.uk/pdbsum) for obtaining its secondary structure, sequence from various organisms are constructed. The rooted
and for obtaining the Ramachandran plot, the SAVES server tree is shown in Figure 1, classified into three clusters by hold-
is utilized. Protein refinement is performed using the ing various sub nodes, showing the primary node of rooted
XELPWV tools and the final model with template structures tree holds all the plasmodium sp, and the other clusters holding
are visualized through chimera tools [24–26]. For understand- the other organism. From this tree, we conclude that the
ing the uncompetitive inhibitors binding region, the CastP adenylosuccinate lyase is more conserved in the plasmodium
(Computed Atlas of Surface Topography of proteins) server family, while other organisms have similar proteins, but not
for locating the binding sites is used. The prepared protein unique with the plasmodium family.
file is subject to input in the CastP server and the output
regions are noted [27].
Homology modeling
The lack of protein structure forced us to construct the pro-
Molecular docking calculations
tein model for adenylosuccinate lyase from P. falciparum.
The 3 D structures of reactant and product are downloaded Since the template is showing 63.91% of identity and 97%
from the PubChem database and for the docking studies, similarity, the adenylosuccinate lyase from P. vivax (2QGA_B
the AutoDock – automated tool for molecular interaction is chain) is considered for the homology modeling. The quality
used [28,29]. The algorithm finds the apt place for the small factors of the modeled protein are validated by comparing
molecules to bind with the macromolecules in the possible with template structure using the procheck and shows that
poses [30]. Initially, the auto grid determines the positional the similar PhiPsi distribution, stereo chemical and energetic
place of the catalytic site through matching the template properties between the target and template protein. The
protein, where AMP binds, and the particular position is modeled structure shows similar topology with the template
 JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION 3

Figure 1. Phylogenetic tree visualization of various organisms with adenylosuccinate lyase.

structure as shown in Figure 2(a,b,c). The model structure bulges, 6 strands, 26 helices, 46 helix-helix interacs, 29 beta
exactly matches with template protein in all the regions and turns and 3 gamma turn. This clearly shows, even if the
0.28 Å RMSD between the template and target protein shows model protein is the matched template structure, there have
the closest match as shown in Figure 2(d). There are some been few structural changes. For analyzing those changes,
loop regions in the template protein, which show missing the Ramachandran plot is performed for the template
residues and those regions are filled in the model protein. (Figure 3(a)) and target protein structure (Figure 3(b)).
Along with this, the whole protein structure of both tem- The Ramachandran plot of template protein shows that
plate and target structure is examined and there have been 91.7% of the residues are in the allowed region, 8.0% of the
few changes in the secondary structure as shown in residues are in the additional allowed region, 0.2% in gener-
Supplementary Figure S2. The secondary structure shows ously allowed region, and there are no residues in disallowed
there are some order to disorder and disorder to order region. While the Ramachandran plot of target modeled protein
changes in the modeled protein, while compared with tem- shows that 92.8% of the residues are in allowed region, 6.3% of
plate structure. The template protein structure shows 3 the residues are in additionally allowed region, 0.5% in gener-
sheets, 1 beta alpha beta unit, 2 beta hairpins, 1 beta bulge, ously allowed region, and there are 0.5% residues in disallowed
6 strands, 27 helices, 47 helix-helix interacs, 24 beta turns region. These comparative results with template and target
and 3 gamma turn. While the target modeled protein shows sequence indicate that the backbone dihedral angles w and u
3 sheets, 1 beta alpha beta unit, 2 beta hairpins, 2 beta
4 E. S. AL-MALKI

Figure 2. (a) Homology modeled adenylosuccinate lyase from P. falciparum with surface. (b) Homology model protein of adenylosuccinate lyase from P. falciparum
(c) Template structure of adenylosuccinate lyase from P. vivax. (d) Molecular matching of adenylosuccinate lyase from P. falciparum (Blue) and P. vivax (orange).

Figure 3. Ramachandran plot of template protein (a) and the target modeled protein (b).

in the model are reasonably accurate in comparison with tem- mechanism kinetic mechanism. The reaction pathway involves
plate protein structure. cleavage of adenylosuccinate to AMP and fumarate and exam-
ining these complexes is core important to understand the
structural aspect of adenylosuccinate lyase. In forward reaction,
Molecular interactions of reactant and product with
the adenylosuccinate is cleaved into adenosine monophosphate
adenylosuccinate lyase
and fumarate as shown in Figure 4. Since, there have been sev-
Adenylosuccinate lyase is a housekeeping gene that is present eral studies performed to understand the reaction mechanism
in many organisms, especially in the plasmodium family. It plays of adenylosuccinate lyase, and as of now, the structural insights
a crucial role in cellular replication, purine nucleotide cycle. of those complexes are not well studied. By this, we examined
Recent studies on Plasmodium falciparum have suggested that the docked complex of adenylosuccinate bound protein and
the C–N bond cleavage is the rate-limiting step, through uni-bi AMP with fumarate bound protein (Figure 5). The 2 D
 JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION 5

Figure 4. Catalytic reaction mechanism of adenylosuccinate lyase utilizing the adenylosuccinate into AMP and fumarate.

Figure 5. (a) Complex structures of adenylosuccinate bound protein, while the adenylosuccinate surface is mapped in red color and (b) AMP with fumarate bound
protein, while the surface of AMP in orange color surface and fumarate is in the pink color surface.

interactions of adenylosuccinate bound protein and AMP with Molecular dynamics simulations
fumarate bound protein are provided in Supplementary Figure
The heat map for the reactant and product shows a similar
S2. The protein amino acids Asp92, Glu97, Ser125 and Arg338
Cross-Correlation Map, due to the same protein and different
shows direct hydrogen bonding interactions with adenylosucci-
ligand features. A single bond broken to form the AMP and
nate (reactant), while in the product, the amino acids Asn90,
fumarate does not make any changes in the heat map. The
Asp92, Gln250, Arg338, Ser343, Arg347 are directly interacting
detail changes can be noted by performing the molecular
with AMP through hydrogen bonds and the amino acids His91,
Lys94, Glu97, His120, Ser125 are directly interacting with fumar- dynamics simulations and for that, the whole complex of
ate through hydrogen bonds. Dynamical Cross-Correlation Map reactant and product is subjected to MD simulations for the
(DCCM) predicted by dynamut is provided in the heat map for timescale of 30 ns. Changes from each snapshot are noted
the reactant and product of adenylosuccinate lyase complex in and the graph for the RMSD is plotted in Figure 7(a,b). The
Figure 6(a,b). Here the Ser 125 is playing the dual role of pro- MD simulation of reactant shows the adenylosuccinate
ton acceptor and donor in the reaction transfer mechanism bound complex is initially flexible and raises up to 10 Å devi-
and the Glu 250 and Glu97 provides marginal base support as ation till the 8th ns. But after the 8th ns, the adenylosuccinate
reaction activator. While in the reactant, the His amino acid as stabilizes inside the binding pocket and the deviations are
electrostatic stabilizer does not play any direct role, but the getting lowered and reaches the ! 7.5 Å. After the 9th ns,
His120 comes forward to hold the fumarate and plays the there is a stable conformation is viewable and till the end of
imperial role in separating the fumarate form the adenylosucci- 30th ns, the adenylosuccinate bound complex is showing the
nate. The other amino acids like Lys, Arg, Asn are contributing stable with the Average RMSD of !7.8 Å RMSD. While the
to the binding and transfer of reactant into product with the reaction mechanism is succeeded and the adenylosuccinate
intermediate steps. is converted into AMP and fumarate, the complex is showing
high deviations from the adenylosuccinate bound complex
6 E. S. AL-MALKI

Figure 6. Dynamical Cross-Correlation Map (DCCM) predicted by dynamut for the reactant enzyme with adenylosuccinate (a) and the product with AMP and
fumarate (b).

Figure 7. RMSD plot for the time scale of 30 ns MD simulation with reactant (a) and product (b) Molecular Dynamics Simulations.

as represented in Figure 7(b). We can be able to see huge Leu200, Lys201, Asn202, Ile203, Glu204, Glu237, Phe240,
ups and downs in the RMSD plot, and the whole simulated His243, Lys470, and Asn471 are having the tendency to
complex does not show any significant stability. We believe adopt the uncompetitive inhibitors for the inhibition of
this could be due to allocation of binding site charge trans- adenylosuccinate lyase. Future compounds, that adopts to
fer that occurs in fumarate removal from the this pocket may have the ability to interact with these spe-
adenylosuccinate. cific amino acids are uncompetitive inhibitors, and those
inhibitors may also combine with competitive inhibitors for
the purpose of combination drug therapy.
Site for uncompetitive inhibition
The whole study reporting the co-factor binding leads to
Conclusion
identify the competitive inhibitors, and in addition, the other
regions for the uncompetitive inhibitors binding also identi- Malaria is a disease caused by the intracellular, protozoan
fied. CastP server predicts several regions, and the top region parasites of the genus Plasmodium and through this work,
of ligand binding site provided in Figure 8. The CastP pre- the plasmodium falciparum enzyme structure of adenylosuc-
dicts the amino acids Leu135, Thyr138, Ile141, His142, Ile146, cinate lyase is examined with react and product. The reaction
 JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION 7

Figure 8. Contours in the protein surface indicated the Active site predicted using the CastP 3.0 server.

mechanism of the adenylosuccinate catalytic mechanism [4] Cui L, Mharakurwa S, Ndiaye D, et al. Antimalarial drug resistance:
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Acknowledgments Mycobacterium smegmatis and Mycobacterium tuberculosis pro-
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The authors thank Deanship of Scientific Research at Majmaah
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University, Al Majmaah, Saudi Arabia for supporting this work.
[14] Ducati RG, Namanja-Magliano HA, Schramm VL. Transition-state
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No potential conflict of interest was reported by the author(s). enzyme with dual activity in the de novo purine biosynthetic
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