Browsing by Author "Nelapati, A.K."

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    A Computational Investigation on Immunogenicity of Uricase from Aspergillus Flavus and Candida Utilis
    (Iranian Chemical Society, 2023) Meena, S.K.; Nelapati, A.K.
    Uricase is a therapeutic enzyme that has applications in adjuvant chemotherapy and the management of treatment-resistance hyperuricemia. It is a key element in gout treatment, a type of arthritis affecting humans due to increased serum uric acid levels. Two available formulations of uricase from Bacillus fastidious and Arthrobacter globiformis were characterized by their high immunogenicity, resulting in the drug's inactivation and hypersensitivity reactions in many patients. This research focuses on protein engineering to find a substitute to PEGylated enzymes with less immunogenicity as an intrinsic characteristic of the protein. We used in silico techniques to spot and modify epitope areas of uricase from Aspergillus flavus (Af) and Candida utilis (Cu), and to decrease the immunogenicity. Both Uricase B-cell epitopes were predicted using surface accessibility and hydrophilicity. Mutations were made to the hot-spot residues to diminish the epitope's antigenicity. Also, molecular docking was used to examine the effect of mutation on uricase activity and stability. Immunoinformatic analysis was done to clarify the structural aspects of the immunogenicity of uricase. For this purpose, the prognostication of immunogenic and allergenic epitopes in uricase structure was performed by using immunogenic peptides relative frequency. To the best of our knowledge, this is the first report of an in silico investigation aimed at decreasing the immunogenicity of uricase from Aspergillus flavus and Candida utilis. © 2023, Physical Chemistry Research. All Rights Reserved.
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    An Approach to Increase the Efficiency of Uricase by Computational Mutagenesis
    (Iranian Chemical Society, 2023) Nelapati, A.K.; Meena, K.S.R.
    Uricase is widely used to treat hyperuricemia and gout. Its clinical use is limited due to side effects such as severe allergy, hypersensitivity, and anaphylactic reactions in some patients. Uricase from Arthrobacter globiformis (Ag) and Bacillus fastidious (Bf) was chosen to improve enzyme binding energy by reducing the deleterious effects in treatment. To reduce the adverse effects of uricase, enzyme should be modified. For this purpose, we performed in silico mutagenesis on uricase. We altered the active site of amino acids of uricase from both sources using f PyMOL. The ligand uric acid was docked with mutated uricase using Autodock 4.0. It was found that mutation of Val64 with Alanine in Ag uricase, and mutation of Gly42 with Isoleucine in Bf uricase improved the binding energy of the enzyme up to 50%. The binding affinity of native Ag uricase docked with uric acid was -8.414 kcal mol-1, while for the mutated enzyme, it was -8.570 kcal mol-1. Binding energies for Bf uricase were -5.221 and 5.389 kcal mol-1 for native and mutated enzymes, respectively. We showed that our in silico model with improved uricase binding energy can facilitate making a potent drug by protein mutagenesis, leading to a drug development with minimum adverse effects to treat hyperuricemia © 2023. Physical Chemistry Research.All Rights Reserved.
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    Computational analysis of therapeutic enzyme uricase from different source organisms
    (2020) Nelapati, A.K.; Ponnanettiyappan, J.
    Background: Hyperuricemia and gout are the conditions, which is a response of accumulation of uric acid in the blood and urine. Uric acid is the product of purine metabolic pathway in humans. Uricase is a therapeutic enzyme that can enzymatically reduces the concentration of uric acid in serum and urine into more a soluble allantoin. Uricases are widely available in several sources like bacteria, fungi, yeast, plants and animals. Objective: The present study is aimed at elucidating the structure and physiochemical properties of uricase by insilico analysis. Methods: A total number of sixty amino acid sequences of uricase belongs to different sources were obtained from NCBI and different analysis like Multiple Sequence Alignment (MSA), homology search, phylogenetic relation, motif search, domain architecture and physiochemical properties including pI, EC, Ai, Ii, and were performed. Results: Multiple sequence alignment of all the selected protein sequences has exhibited distinct difference between bacterial, fungal, plant and animal sources based on the position-specific existence of conserved amino acid residues. The maximum homology of all the selected protein sequences is between 51-388. In singular category, homology is between 16-337 for bacterial uricase, 14-339 for fungal uricase, 12-317 for plants uricase, and 37-361 for animals uricase. The phylogenetic tree constructed based on the amino acid sequences disclosed clusters indicating that uricase is from different source. The physiochemical features revealed that the uricase amino acid residues are in between 300-338 with a molecular weight as 33-39kDa and theoretical pI ranging from 4.95-8.88. The amino acid composition results showed that valine amino acid has a high average frequency of 8.79 percentage compared to different amino acids in all analyzed species. Conclusion: In the area of bioinformatics field, this work might be informative and a stepping-stone to other researchers to get an idea about the physicochemical features, evolutionary history and structural motifs of uricase that can be widely used in biotechnological and pharmaceutical industries. Therefore, the proposed in silico analysis can be considered for protein engineering work, as well as for gout therapy. 2020 Bentham Science Publishers.
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    Computational analysis of therapeutic enzyme uricase from different source organisms
    (Bentham Science Publishers P.O. Box 294 Bussum 1400 AG, 2020) Nelapati, A.K.; JagadeeshBabu, J.
    Background: Hyperuricemia and gout are the conditions, which is a response of accumulation of uric acid in the blood and urine. Uric acid is the product of purine metabolic pathway in humans. Uricase is a therapeutic enzyme that can enzymatically reduces the concentration of uric acid in serum and urine into more a soluble allantoin. Uricases are widely available in several sources like bacteria, fungi, yeast, plants and animals. Objective: The present study is aimed at elucidating the structure and physiochemical properties of uricase by insilico analysis. Methods: A total number of sixty amino acid sequences of uricase belongs to different sources were obtained from NCBI and different analysis like Multiple Sequence Alignment (MSA), homology search, phylogenetic relation, motif search, domain architecture and physiochemical properties including pI, EC, Ai, Ii, and were performed. Results: Multiple sequence alignment of all the selected protein sequences has exhibited distinct difference between bacterial, fungal, plant and animal sources based on the position-specific existence of conserved amino acid residues. The maximum homology of all the selected protein sequences is between 51-388. In singular category, homology is between 16-337 for bacterial uricase, 14-339 for fungal uricase, 12-317 for plants uricase, and 37-361 for animals uricase. The phylogenetic tree constructed based on the amino acid sequences disclosed clusters indicating that uricase is from different source. The physiochemical features revealed that the uricase amino acid residues are in between 300-338 with a molecular weight as 33-39kDa and theoretical pI ranging from 4.95-8.88. The amino acid composition results showed that valine amino acid has a high average frequency of 8.79 percentage compared to different amino acids in all analyzed species. Conclusion: In the area of bioinformatics field, this work might be informative and a stepping-stone to other researchers to get an idea about the physicochemical features, evolutionary history and structural motifs of uricase that can be widely used in biotechnological and pharmaceutical industries. Therefore, the proposed in silico analysis can be considered for protein engineering work, as well as for gout therapy. © 2020 Bentham Science Publishers.
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    In silico structural and functional analysis of bacillus uricases
    (Bentham Science Publishers, 2021) Nelapati, A.K.; Meena, S.; Singh, A.K.; Bhakta, N.; JagadeeshBabu, P.E.
    Background: Excluding humans, the peroxisomal uricase is responsible for the catabolism of uric acid into allantoin in many species like microorganisms, plants, and inverte-brates. Particularly in humans, the synthesis and excretion of uric acid are naturally balanced. When the uric acid concentration crosses 7 mg/dl, it results in conditions such as hyperuricemia and gout. Uricase is one of the potential sources for the reduction of uric acid in humans. Uricase is also widely used as a commercial diagnostic reagent in medical and clinical biochemistry to esti-mate the uric acid concentration in blood and other biological fluids. Computational approaches can be used for screening and investigation of uricase enzyme with desirable characteristics that can be employed in diverse industrial applications. Objectives: The present study deals with computational-based structural, functional, and phylogenetic analyses of uricase enzymes from various Bacillus species. Methods: Seventy uricase protein sequences from Bacillus species were selected for multiple sequence alignment, phylogenetic analysis, motif assessment, domain architecture examination, understanding of basic physicochemical properties and in silico identification of the composition of amino acids in uricase. Further, structural (secondary and tertiary structure prediction), and functional (CYS_REC, MOTIF scan, CD-search, STRING, SOSUI, and PeptideCutter) analyses of uric-ase were performed. Results: Bacillus simplex (WP_063232385.1) was chosen as the representative species of the Bacillus genera. The three-dimensional (3D) structure of B. simplex uricase was predicted and validated using QMEAN, RAMPAGE, ERRAT, Verify 3D and PROQ servers. The analysis revealed that the tertiary structure of the selected uricase has good quality and acceptability. Conclusion: Computational analysis of uricase from various Bacillus sources revealed that all the selected Bacillus uricases are active within acidic to a neutral environment, and thermally stable with a molecular weight ranging from 35.59-59.85kDa. The secondary structure analysis showed that all uricases are rich in alpha-helices and sheets. The CDD tool identified two conserved do-mains, one of which belongs to OHCU decarboxylase and another belongs to Uricase superfamily. The quality estimation of 3D modeled protein gave a high overall quality factor score of 94.64. Al-so, all Bacillus species of uricase enzyme and their corresponding genes showed a strong correlation from the phylogenetic comparison of the selected taxa. The present detailed computational investigation on the uricase protein could help in screening a suitable uricase producing microbe with desirable characteristics for industrial application. © 2021 Bentham Science Publishers.
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    In-silico epitope identification and design of Uricase mutein with reduced immunogenicity
    (2020) Nelapati, A.K.; Das, B.K.; Ponnan, Ettiyappan, J.B.; Chakraborty, D.
    The clinical utilization of Uricase against gout is limited due to the immunogenicity. In the present article, we identified the antigenic determinants of Uricase and reduced their immunogenicity via in-silico mutagenesis. Multiple sequence alignment and motif analysis were carried out to identify the conserved residues in evolutionary process. Emini surface accessibility, Parker hydrophilicity, and Karplus & Schulz flexibility methods were employed to predict the linear B-cell epitopes of both Ag-Uricase and Bf-Uricase. Deimmunization approach identified T-cell epitopes and the hot spot residues. Reduced antigenic probability was obtained in case of T159W, D169C, N264W and Y203D mutations for Ag-Uricase, while S139 V, K215W, G216 F and I172 P mutations for Bf-Uricase. The binding affinity values of uric acid towards the catalytic pocket of Ag-Uricase and Bf-Uricase models were found to be -48.71 kcal/mol and -40.93 kcal/mol, respectively. This energy is further stabilized in the mutant model by -6.36 kcal/mol and -1.45 kcal/mol for Ag-Uricase and Bf-Uricase, respectively. About 100 ns molecular dynamics simulation was performed to evaluate the conformational stability of both native and mutated Uricase. Insights obtained from this study provide guidelines for experimental design of Uricase muteins with reduced antigenicity. 2020 Elsevier Ltd
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    In-silico epitope identification and design of Uricase mutein with reduced immunogenicity
    (Elsevier Ltd, 2020) Nelapati, A.K.; Das, B.K.; JagadeeshBabu, J.B.; Chakraborty, D.
    The clinical utilization of Uricase against gout is limited due to the immunogenicity. In the present article, we identified the antigenic determinants of Uricase and reduced their immunogenicity via in-silico mutagenesis. Multiple sequence alignment and motif analysis were carried out to identify the conserved residues in evolutionary process. Emini surface accessibility, Parker hydrophilicity, and Karplus & Schulz flexibility methods were employed to predict the linear B-cell epitopes of both Ag-Uricase and Bf-Uricase. Deimmunization approach identified T-cell epitopes and the hot spot residues. Reduced antigenic probability was obtained in case of T159W, D169C, N264W and Y203D mutations for Ag-Uricase, while S139 V, K215W, G216 F and I172 P mutations for Bf-Uricase. The binding affinity values of uric acid towards the catalytic pocket of Ag-Uricase and Bf-Uricase models were found to be -48.71 kcal/mol and -40.93 kcal/mol, respectively. This energy is further stabilized in the mutant model by -6.36 kcal/mol and -1.45 kcal/mol for Ag-Uricase and Bf-Uricase, respectively. About 100 ns molecular dynamics simulation was performed to evaluate the conformational stability of both native and mutated Uricase. Insights obtained from this study provide guidelines for experimental design of Uricase muteins with reduced antigenicity. © 2020 Elsevier Ltd