Computational analyses of RPIA gene mutation causing Ribose-5-phosphate isomerase deficiency: a rarest known metabolic disorder in humans

Abstract

The main aim of current study was to perform in silico functional analysis of all reported RPIA mutations. In silico analysis was done using different tools i.e. trRosetta tool, Autodock Vina, PatchDock and iMODS. Among all the reported mutations, lowest percentage identity of 4.69% with wild-type protein was shown by splice site mutation, while highest percentage identity of 78.78%, was shown by p.Ser61Val mutation. In protein-substrate docking, wild-type protein was docking with substrate with 11 different bonds. However in case of mutant protein highest interaction with substrate molecule was shown by mutant p.Ile257Thr proteins via 13 different bonds, while lowest interaction was noted in mutant p.Asn255Ilefs17Term protein via 5 bonds. From the results, it was concluded that the binding of RPIA with ribose 5-phosphate is altered by the type of amino acid substitution, as well as the nature and number of bonds, which are capable to influence its biological activity.

KEYWORDS:

• Ribose-5-phosphate
• recessive
• metabolic
• In silico

1. Introduction

Ribose-5-phosphate isomerase (Rpi: EC 5.3.1.6), an aldose-ketose isomerase enzyme, operates in the pentose phosphate pathway (PPP) and catalyzes the isomerization of ribulose-5-phosphate (Ru5P) to ribose-5-phosphate (R5P) and vice versa. Although a few organisms have fully or partially nonfunctional pentose phosphate pathways [1], it appears that Rpi is a universal enzyme present in all life forms and that all organisms require a mechanism for the interconversion of these two sugar phosphates. The expression of RPI occurs in two analogous enzymatic forms i.e. RPIA and RPIB. These forms are very much dissimilar in structure and evolutionary route but in terms of function, they both are catalytic in nature. RPIA is common among all taxanomic groups, while RPIA is ubiquitous in bacteria and lower eukaryotes [2,3]. RPIA gene has a single transcript consisting of a single Ribose-5-phosphate isomerase enzymatic domain from amino acid 56 to 308 [2].

Ribose-5-phosphate isomerase deficiency (RPID, MIM# 608611) belongs to the group of extremely rare metabolic autosomal recessive genetic disorders. RPID is caused by pathogenic mutations in the RPIA gene [4], which encodes for ribose-5-phosphate isomerase enzyme, a key regulator in the non-oxidative phase of the pentose phosphate pathway. In this diseased condition, the pentose phosphate pathway (PPP) is disturbed and the inter-conversion between two sugars (ribulose 5-phosphate and ribose 5-phosphate) doesn’t take place properly. The results in the progressive condition of leukodystrophy associated with polyol metabolism defects can be observed [4].

Van der Knapp and co-workers reported the first case of Ribose-5-Phosphate isomerase deficiency in 1999. They documented a 14-year-old boy with delayed development, insidious psychomotor regression, progressive leukoencephalopathy, epilepsy and defective polyol metabolism of unknown aetiology [5]. As very little is known about this disorder its exact prevalence is still unknown. So far, scientific literature and the human genome mutation database (HGMD) have enlisted only 10 mutations in the RPIA gene in different ethnicities of the world to cause Ribose-5-Phosphate isomerase deficiency. Among reported mutations, eight are missense/nonsense, one is small deletion and one mutation is splice site mutation (Figure 1(a)).

Figure 1. (a) The RPIA gene exon and its exon, (b) RPIA protein’s largest active site pocket and (c) Protein substrate docking of RPIA protein with its substrate ribose-5-phosphate molecule.

Ribose-5-Phosphate isomerase deficiency results in the build-up of pentoses and pentose phosphatases and elevated rate of ribitol and arabitol in brain and body fluids by rendering them as the metabolic end product. Researchers presume that the accumulation of Rpi may disturb the activity of different chemicals in the body that act as neurotransmitters (transmit signals between neurons in the brain), resulting in an increased possibility of different phenotypes such as intellectual disability [4,6].

The current in silico study was intended to investigate and compare the functional influence of all reported RPIA mutations on the activity of Ribose-5-phosphate isomerase enzyme.

2. Materials and methods

2.1. Pathogenicity of Splice site variant

Splicing effects of genetic variation (RPIA, c.347-1G > A) were measured by utilizing a prediction tool called JSI splice site. This tool is found as varSEAK Online and varSEAK software. Information on canonical splice sites (core motif GT for 5′ donor splice sites or AG for 3′ acceptor splice sites) 19 is required by this software [7].

Once the variant pathogenicity was identified, the protein changes were predicted and translated through the online tool ExPASy (Expert Protein Analysis System) translation tool (https://web.expasy.org/translate/). ExPASY assisted in the translation of protein based on the provided genomic sequence predicted through the varSEAK tool. Different frames of mutant protein were predicted by ExPASY, out of which the longest frame with the longest amino acids sequence was selected for the present study.

2.2. Structural modelling

HGMD list was retrieved for all the described variants of RPIA, whereas the protein sequence for the wild-type RPIA was obtained from Ensemble Genome Browser (https://asia.ensembl.org/index.html, accessed on 1 July 2022). 3D protein structural model prediction was done by using the trRosetta online tool [8]. Among the predicted models, those models were selected that had the highest CS (confidence score). trRosetta, an algorithm for quick and precise prediction of protein structure, generates protein structure based on direct energy minimizations with a restrained Rosetta. The restraints include inter-residue distance and orientation distributions, predicted by a deep neural network.

These 3D models were visualized by using the Chimera tool [9]. The model was employed to superimpose the wild-type and the mutant version of protein individually to examine the differences among them in the form of percentage identity. A close functional interactor of RPIA was determined using the STRING online tool [10] to be an RPE protein. 3D models were verified using Ramachandran plots and results are provided as supplementary files.

2.3. Protein-substrate docking

Protein-substrate docking for normal and all mutant PRIA proteins with ribose 5-phosphate molecule was done using the online tool Autodock Vina and Vina MGL tool [11] and the protein-substrate docked complexes were analysed through Discovery Studio 2020 [12]. Wild-type RPIA protein-active site pockets were predicted through the online tool CASTp [13]. More help was taken from the following literature [14–17].

2.4. Protein-Protein interaction

The wild-type and mutant RPIA proteins were examined for protein-protein interaction by using the Patch dock tool (available at https://bioinfo3d.cs.tau.ac.il/PatchDock/), a program designed for molecular docking. The input data includes two molecules of any kind: peptides, DNA, proteins, or drugs. It generates output in the form of a list of potential complexes classified by shape complementarity criteria. The interactions were visualized by using the LigPlus tool to examine the differences between the normal interactions in comparison with the mutant proteins [18].

2.5. Protein simulations

iMODs tool available at www.imods.iqfr.csic.es (accessed on 7 August 2022) was utilized to perform the protein simulations. This tool functions on the principle of normal mode analysis (NMA). Simulation results were calculated in the form of deformability of residues and eigenvalues of the complex keeping the default basic settings.

2.6. Conserved sequence alignment

To check the conservation of all mutated RPIA amino acids, conserved sequence alignment was also performed. Conserved Sequence Alignment was carried through Clustal Omega tools (Embl-Ebi Clustal Omega https://www.ebi.ac.uk/Tools/msa/clustalo/, accessed May 13, 2022). Clustal Omega is a novel multiple-sequence alignment program which operates based on the Hidden Markov Model (HMM) profile-profile techniques and seeded guide trees. Using this program, alignments between three or more related sequences could be generated.

2.7. Pathway analysis of identified protein

Pathway analysis of identified protein was done through KEGG (Kyoto Encyclopedia of Genes and Genomes) and all the pathways flowcharts are adopted from KEGG (https://www.genome.jp/kegg/, Accessed on 1 March 2022).

3. Results

3.1. Mutation information

Information on all the reported RPIA mutation was collected from the literature. The information including cDNA Change, protein change, the exonic position of the mutation on the RPIA gene, ethnicity of the reported mutations and rsIDs are summarized in Table 1. Splice site mutation in RPIA i.e. c.347-1G > A results in complete deletion of exon 3 (Supplementary Figure 1) and results in short truncated function less protein of 128 amino acids (longest frame).

Table 1. All reported RPIA mutations with their respective percentage identity with wild-type RPIA protein.

Mutation	% Identity	Effect on protein	rs ID	Ethnicity	Reference
c.762delG	73.53	p.Asn255Ilefs17Term	rs730880316	Amsterdam, Netherland	[1]
c.404C > T	77.17%	p.Ala135Val	rs121918591	Amsterdam, Netherland	[1]
c.627G > C	78.14%	p.Trp209Cys	rs1558699183	Germany, Turkey, Iran, Egypt, Afghanistan, and Pakistan	[2]
c.347-1G > A	4.69%	Complete deletion of exon 3	rs1422698928	New Brunswick, USA	[3]
c.540delG	67.18%	p.Gln181Argfs14Ter	–	Amsterdam, Netherland	[1]
c.182C > T	78.78%	p.Ser61Val	–	Amsterdam, Netherland	[1]
c.592T > C	77.81%	p. Phe198Leu	–	–	[4]
c.770T > C	78.46%	p.Ile257Thr	–	India	[5]
c.253G > A	78.46%	p.Ala85Thr	rs1399404493	New Brunswick, New Jersey	[3]
c.796C > T	78.46%	p.His266Tyr	rs147431137	-	[6]

3.2. Structural analysis

3D models of wild-type and mutant RPIA protein were designed as shown in Figure 2. Mutant RPIA protein models were individually superimposed with wild-type RPIA proteins, as shown in Figure 3. The analysis showed visible differences in the physical structures, which were documented in the form of percent identity (PI) between wild-type and mutant RPIA proteins. The PI shows the resemblance between the two models. Mutant p.Ser61Val presented the maximum resemblance index to the wild-type RPIA protein with a PI of 78.78%, while the lowest PI was presented by splice site mutant RPIA protein (predicted to be 128 amino acid long protein) mutant with a PI of 4.69%. This PI was obtained by using the Chimera tool. All the percent identities of wild-type and mutant RPIA proteins are listed in Table 1.

Figure 2. 3D models of wild-type and all reported RPIA mutant proteins.

Figure 3. Superimposed structure of all reported RPIA proteins with wild-type RPIA protein.

3.3. Protein-substrate docking

For protein-substrate docking first active site pockets of RPIA protein were determined and found that the largest active site pocket of RPIA protein was present on its enzymatic domain from amino acid 56 to 308. This domain has enzymatic activity and is known as Ribose-5-phophatase isomerase, as shown in Figure 1(b). After active site pocket predication, Protein-substrate docking was done between wild-type and all mutant RPIA proteins with ribose-5-phosphate molecules to check the type of bonding and interacting residues. Wild-type RPIA protein was docked with close substrate molecule with 11 different bonding through 11 different residues, as shown in Figure 1(c). However in the case of mutant protein highest interaction with substrate molecule was shown by mutant p.Ile257Thr proteins via 13 different bonds, while the lowest interaction with substrate molecule was noted in mutant p.Asn255Ilefs17Term protein via only 5 bonds. The summary of interacting residues and interacting bonds of all mutant proteins is given in Table 2 and Figure 4.

Table 2. Protein-substrate interaction of wild-type and all mutant RPIA proteins with substrate ribose-5-Phosphate molecule.

Mutation/variant	Residues attached via Conventional Hydrogen Bond	Residues attached via Van der Waals Force	Residues attached via C -H Bond	Residues attached via Attractive Charge	Residues attached via Unfavourable Donor-Donor or Acceptor-Acceptor Bond	Total no of Bond
Normal/Wild type	Glu182, Ser104, Thr107, Ala162, Asp163	Gly175, Ile108, Gly161, Ser106, Ile195, Lys200	Gly105	Asp160	Lys173	11
p.His266Tyr	Lys82, Thr107, Gly174,Gly175, Glu182	Ser106, Ile195, Ala196, Gly105, Gly161, Lys173	-	Glu182, Asp160, Asp163	-	8
p.Ala135Val	Gly174, Ala162, Lys200, Glu182,	Lys173, Ile108,Ser106, Gly161,Lys82	Gly105	Glu182, Asp160, Asp163	-	9
p.Ser61Val	Asn204, Lys203, Val265, Leu205, Trp268, Lys267, Asn170, Ser269	Asp168, Asn289, Ile288, His266	-	Asp166	-	9
p.Trp209Cys	His266, Trp268, Ser269, Asp166	Lys203, Leu205, Asn204, Lys267	-	Asp166	-	9
p.Gln181Argfs14Term	Ser65, Ser67	Arg118, Gln121, Glu91, Arg114, Ile68, Cys69, Asn66	-		Glu117, Asp64, Arg87	7
p.Ala85Thr	Trp268, Ser269, Asp166	Lys203, His266, Ala167, Lys267, Ile288, Asp168, Val265	Asn204	Asp166	Leu205,Asn170	9
p.Ile257Thr	Asn170, Asn204, Ser269, Trp268, His266	Glu270, Lys203,Leu205, Ile288	Lys 267	Asp166	Val265	13
p.Asn255Ilefs17Term	Pro130, Leu149, Asp148	Ile129, Gln181, Thr131, Ser132, Asp252, Phe133, Arg136		Asp148	-	5
p.Phe198Leu	Thr107, Ala162, Lys173, Gly174, Glu182, Asp163	Ser104,	Lys200, Asp163	Asp163	-	10
Splice site variant	Ser8, Gly5, Asp126	Met1, Pro127, Ile102, Gln2, Arg3, Gln128	Pro4, Gly101	Asp126		6

Figure 4. Protein-substrate docking of all mutant RPIA proteins with substrate Ribose-5-phophase molecules.

3.4. Protein–protein interactions

The interactions between all mutant and wild-type RPIA proteins with close interactor RPE protein were evaluated for type and the number of bonds made for the stable interaction. Interactions were observed to examine the difference between wild-type and mutant protein interaction. For the wild-type RPIA, the interaction was made stable by forming 15 bonds out of which 2 were salt bridges while 13 were H-bonds. The interacting residues of RPIA making these bonds with RPE protein were i.e. Glu154, Arg95, Arg191, Gln48, Arg46, Arg42, His93, Asn92, Thr58, Thr60, and Asn97. Among mutant proteins of RPIA, the highest-level interaction was observed in mutant Asn255Ilefs17Term protein, showing 12 different residues of mutant protein interacting with RPE and interact through 16 bonds i.e. 2 salt bridges and 14 H-bonds, to make the interaction stable. Whereas the lowest interaction is observed in the splice site variant, which interacted with RPE protein by only 3 amino acids through 3 H-bonds. Complete detail of the protein-protein interaction of mutant and wild-type RPIA protein is shown in Table 3.

Table 3. Interaction of normal and mutant proteins of RPIA with close interactor RPE proteins.

Protein	Interacting Residues Of RPIA	Interacting Residues RPE	Type Of Bond
RPIA (wild-type)	Glu154, Arg95, Arg191, Gln48, Arg46, Arg42, His93, Asn92, Thr58, Thr60, Asn97	Arg228, Thr165, Asp136, Ile135, Ser208, Asp205, Glu204, Ser203, Arg202, Ser169, Arg109, Asp64, Asn89, Ala2	2 salt bridges, 13 H-bonds
p.Asn255Ilefs17Term	Asp148, Arg151, Asp150, Arg136, Asn123, Asp64, Asn66, Ser39, Ser65, Cys62, Lys120, Gly143	Arg207, Asp205, Leu27, Ala31, Gln222, Ser169, Asp171, Asn192,Leu170, Lys114, Ser225, Gly4, Ser3	2 salt bridges, 14 H-bonds
p.Ala135Val	Gln134, Gln141,Tyr142, Glu79, Val109, Cys62	Asn211, Ala31, Gln63, Arg25, Asp28, Ala86	6 H-bonds
p.Trp209Cys	Gly176, Asp252, Gln137, Gln134,Gly105, Thr51, Gly53, Arg46, His110	Arg228, Arg224, Glu118, Arg164, Gln166, Lys151, Lys185, Gln126	2 salt bridges, 9 H-bonds
p.Gln181Argfs14Ter	Gln141, Gln137, Gln134, Ser67, Asp64, Tyr142, Lys120, Tyr11, Asn123	Tyr33, Ala2, Met1, Arg207, Arg214, Cys5, Asp32, Asp64, Met201, Gly62	11 H-bonds
p.Ser61Val	Ala71, Glu122, Glu91, Ala16, Arg42, Gly28, Ala22, Pro19, Leu18, Asn66, Val61, Cys62	Gln222, Arg207, Asn211, Arg214, Arg202, Glu26, Asp155, Gln150, Gly149, Leu27, Asp64	2 salt bridges, 11 H-bonds
p.Phe198Leu	Arg151, Glu154, Asp148, Thr60, Asn57, Thr58, Ser33, Gln2, Gly28, His40	Asn13, Gly148, Asn18, Arg202, Glu96, Gly41, Phe43, Arg164, Glu204, Met1, Glu126	2 salt bridges, 9 H-bonds
p.Ile257Thr	Tyr142, Gln121, Lys83, His110, Cys62, Lys120, Asn57, Arg52	Pro180, Glu219, Asp227, Ser225, Glu188, Ser169, Asp136	2 salt bridges, 7 H-bonds
p.Ala85Thr	Gly50, Arg151, Asp150, Gln181, Arg136, Asp252, Asn253	Arg228, Glu188, His184, Glu219, Gln222, Ser3, Ser218	1 salt bridges, 8 H-bonds
p.His266Tyr	Leu144, Thr145, Gly53, Leu146, Gln141, Leu124, Gln137, Gln48, Arg52, Arg42, Arg151,	Arg214, Asp32, Gln222, Asn211, Met1, Asp155, Glu154, Pro145, Asp64,Lys185	2 salt bridges, 10 H-bonds
Splice site variant	Leu125,Gln2,Asp126	Thr48,Asp15,Arg202	3 hydrogen bonds

3.5. Protein normal mode analysis (NMA) simulation

Protein NMA was done on wild-type RPIA and substrate ribose-5-phosphate complex, mutant with highest interaction i.e. p.Ile257Thr, and substrate ribose-5-phosphate complex and mutant with lowest interstation i.e. p.Asn255Ilefs17Term and substrate ribose-5-phosphate complex. The results were calculated in the form of the position of deformability of the protein residues and Eigen values. Eigenvalue is the energy required to break the complex. The more the Eigen value the more energy will be required to break the complex and vice versa. In the case of wild-type RPIA and substrate ribose-5-phosphate complex position of deformable residues was between 150 and 200, which also proves our results in protein-substrate docking and it requires a low Eigen value of 1.2882 × 10⁻⁷. In the case of p.Ile257Thr and substrate ribose-5-phosphate complex, deformable residues were present not only in the enzymatic domain but also in the non-enzymatic domain i.e. before amino acid no 56; however, all the substrate-binding residues were present in the enzymatic domain i.e. amino acid 56 to 308 as shown in Figure 4. Additionally, its Eigen energy was much higher than wild-type protein i.e. 2.976944 × 10⁻⁷. In the case of p.Asn255Ilefs17Term and substrate ribose-5-phosphate complex, the entire mutant protein was completely disturbed and almost entire residues were deformable and it also showed the highest Eigen value of about 5.93708 × 10⁻⁴ (Figure 5).

Figure 5. IMods results of (a) wild-type RPIA protein showing the position of defoamable residues and Eigen value of the complex, (b) Mutant p.Ile257Thr RPIA protein showing the position of deformable residues and Eigen value of the complex and (c) Mutant p.Asn255Ilefs17Term RPIA protein showing the position of deformable residues and Eigen value of the complex.

3.6. Conserved sequence alignment

To check the conservation of all the substituted amino acids, conserved sequence alignment was performed in all the mutant proteins. Conservation of Homo sapiens RPIA protein was done with different species. All the substituted amino acids were highly conserved throughout the species. Complete details of all the substituted amino acids are shown in Figure 6.

Figure 6. Conserved sequence alignment of the RPIAA protein showing the conservation of the reported substituted amino acids.

3.7. Pathway analysis

Ribose-5-phosphate isomerase is a metabolic enzyme and is involved in three major pathways i.e. Pentose phosphate pathway, carbon metabolism pathway, and biosynthesis of amino acids. Schematic diagrams of all the said pathways are shown in Supplementary Figures 2–4.

4. Discussion

The Human RPIA (ribose 5-phosphate isomerase) gene (OMIM#180430) is a protein encoding gene, present on cytogenetic locus 2p11.2. The human RPI gene consists of 9 exons having an estimated size of 59 Kb with orientation on the plus strand and encodes a protein monomer that extends to 311 amino acid residues. RPIA gene has a single transcript consisting of 13 domains and is associated with 14527 variant alleles (EMBL-EBI release 107 – Jul 2022). RPIA expression has been confirmed in multiple tissues including the bone marrow, placenta, brain, heart, kidney, lung, colon, lymph node, and testis [3].

A study conducted by Spensor and Hopkinson (1980) established ribose 5-phosphate as a dimer and determined by a single structural locus. RPIA is the key factor in nucleotide metabolism and regulates the synthesis of nucleic acids i.e. RNA and DNA. Ribose and its metabolic product deoxyribose, provide the structural sugar components of nucleic acids and play a major role in cells undergoing division. Ribose is an integral part of the structural assembly of cellular intermediates such as coenzyme A, FAD, NADP⁺, NADPH⁺, AMP, ADP, and ATP [19,20].

Extensive studies showed that RPIA (MIM# 180430) being a major regulator in the PPP non-oxidative pathway, is associated with the onset of carcinogenesis by generating nucleotides essential for nucleic acid synthesis in rapidly dividing cancer cells [21]. PPP is a key factor in promoting tumour by facilitating cancerous cells with bio-materials and energy required for their rapid growth [22]. A study intended to explore the potential role of RPIA in colorectal cancer (CRC) showed over-expression of RPIA in patients and the zebrafish transgenic model. Oncogencity is induced in colon cancer cell lines by β-catenin activation facilitated by RPIA. The results demonstrated that the RPIA gene C-terminus (comprising amino acids 290 to 311) is essential in RPIA-regulated tumorigenesis [23]. RPIA showed significant involvement in hepatocellular carcinoma (HCC) via PP2A and ERK (extracellular signal-regulated kinase) signalling. Tumour growth in animal models (nude mice) was enhanced by elevated RpiA levels, inducing cell proliferation and colony formation [24]. For the increased synthesis of nucleotides, cancer cells redirect glytolytic flux into the PPP nonoxidative route to form ribose 5-phosphate by RpiA [25,26]. RPIA gene deregulation is also a key factor in promoting tumour formation in lung and breast tissues [25]. Over-expression of RPIA speeds up the proliferation of lung cancer cells. The knockdown model of RPIA demonstrated enhanced ROS levels leading to autophagy, apoptosis, and cellular senescence in lung cancer cells and a decrease in tumour growth by halting cell proliferation [24]. Szwarc and team (2018) found evidence of RPIA involvement in endometrial cancer, a fourth common tumour in women belonging to United States ethnicity with a high malignancy rate [27].

Malfunctions at the genetic level include the incorporation of pathogenic variants in the RPIA gene that can alter its normal function and lead to ribose 5-phosphate isomerase deficiency (RPIAD, MIM# 608611). This condition is characterized by the accumulation of pentoses and pentose phosphatases and an elevated rate of ribitol and arabitol in the brain and body fluids by rendering them as the metabolic end product. RPID is induced by an autosomal recessive mutation (OMIM) and is classified as a metabolic disease. It is considered the rarest among the three enzyme defects of PPP [5,28]. It manifests progressive leukoencephalopathy associated with abnormalities of polyol metabolism [5] and leukodystrophy [29,30].

A current in silico study was performed on reported RPIA gene mutations causing RPID. The effect on splice site mutation was determined which confirmed the findings of Brooks et al., 2018 that the splice site change results in complete loss of exon 3 of the RPIA protein [6]. The percentage identity of all the models was much lesser not only in the frameshift mutation but also in the missense mutation, (Figures 2 and 3). Multiple sequence alignment also confirms that all the mutated amino acids were present in the highly conserved region of the PRIA protein (Figure 6). Conserved sequence alignment and protein modelling confirm the results of previous studies. Hijikata and colleagues reported a major structural position of autosomal recessive mutations as [29] the buried region of a protein. Protein structure is also affected and destabilized by these mutations. As the protein’s structural and functional alterations due to the substitution of an amino acid were difficult to accurately predict, a Bayesian hierarchical model was used to investigate the missense mutation’s disease causality [30]. Agrahari et al confirmed the effect of missense mutations on the stability and dynamics of the proteins and induction of disease phenotypes by mutations in patients [31].

Similarity index of all reported mutations were noted and among them Splice site mutation c.347-1G > A showed the lowest similarity index with wild-type RPIA protein. This result also confirms the finding of previous studies that the majority of the splice site mutations resulting in human health defects, incorporate the invariant GT and AG dinucleotides in the 5′ and 3′ splice sites [32,33]. Exon delineation and precise splicing are determined by these dinucleotides. However, mis-splicing can also occur through mutations taking place at other locations of the 5′ or 3′ splice site and results in exon skipping, intron retention or cryptic splice site activation.

Wild-type RPIA protein was docking with substrate Ribose-5-phosphate molecule with 11 different bonds while among the mutant proteins highest interaction with the substrate molecule was shown by p.Ile257Thr via 13 bonds, while the lowest interaction was shown with the substrate molecule by p.Asn255Ilefs17Term protein. So we examined the complexity of these protein complexes. Analysis of these proteins through normal mode analysis found that the interaction of p.Ile257Thr was greater with the substrate molecule due to the deformability of the residues in the protein due to mutation; however, the resulting complex formed (p.Ile257Thr and substrate complex) was of high energy compared to wild-type and substrate complex. This high-energy molecule cannot be broken down easily which is against the actual action of the enzyme, which lowers the activation energy. The same situation was with mutant p.Asn255Ilefs17Term where the energy of the complex was even higher. We also know that enzymes play an indispensable part in lowering the activation energy of reactions that is, the amount of energy required for the reaction to initiate. Enzymes regulate reactions by binding to substrate molecules and holding them in a position where the chemical bond breakage and formation processes take place more readily [34].

Moreover, apart from the results we suggested that early anamnesis and treatment may potentially reduce the progression of the disease. In this regard and uncertainty associated with the various phenotypes of metabolic anomalies, a comprehensive neurological checkup must be a compulsory part of regular pediatric appointments [34,35]. Radiological tests such as CT and MRI scans, could help detect patients who are likely to have congenital metabolism mutations [36–39].

5. Conclusion

The present computational study was done to check the consequences of all RPIA mutations reported so far, on interactions of ribose-5-phosphate with its respective protein by exploiting various modelling and docking methods. The results revealed that the type of amino acid substations, number of the bonds, and deformability of the amino acids after the mutation alter the binding and dissociation of the ribose-5-phosphate molecule with the RPIA enzyme, leading to an alteration of its action. Furthermore, it was also concluded that the intensity of damage to RPIA differs with the type of mutation. No related study on the RPIA gene has been reported so far, making this research the first in silico insight on all documented mutations of RPIA, which can present a better and more inclusive understanding of the topic to scientists and researchers in the field of genetics and metabolic disorders.

Authors contribution

All authors contributed equally.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The computational data are stored in the password-protected personal computers of M.M., which are available upon request.