Homology modeling of microbial Nattokinase enzyme, An Anti-blood clotting (Fibrinolytic) agent using computational tools

 

Praveen Reddy P.

Department of Microbiology, Vivekananda Degree and PG College, Karimnagar-505001, Telangana, India.

*Corresponding Author E-mail: biomicroppr@gmail.com

 

ABSTRACT:

Many people are suffering from cardiovascular diseases and some may die due severe heart strokes. The main reason for heart attacks is clots in the circulating blood due to fibrin. The Nattokinase enzyme which is produced by certain microbes especially Bacillus species is an effective drug for preventing cardiovascular disorders and other complications. The Nattokinase degrades fibrin and removes blood clots. The administration of Nattokinase had minimized the blood clots in patients. In the present paper the sequence of Nattokinase enzyme was derived from UniProt web-server and used to develop its three-dimensional model in Phyre2 server. The quality of developed model structure of Nattokinase enzyme was validated using the most widely used web programs Verify3D and ProSA. Further an improved Nattokinase enzyme can developed using various advanced bioinformatics tools. Such improved Nattokinase enzyme can be produced in the laboratory by microbes.

 

KEYWORDS: Cardiovascular diseases, heart strokes, Nattokinase, blood clots, UniProt, Phyre2, Verify3D and ProSA.

 

 


1. INTRODUCTION:

Nattokinase is regarded as an effective fibrinolytic enzyme present in Japanese food Natto. The Natto is a fermented food obtained by the activity of Bacillus subtilis on soy beans. Nattokinase enzyme is a potent fibrinolytic agent. Nattokinase clears clots in the blood and reduces the risk of heart strokes. Natto is consumed to reduce the risk of cardiovascular related disorders as it contains Nattokinase. Even the purified Nattokinase is also consumed as a drug. The Nattokinase enzyme degrades blood clots by breaking fibrin and plasmin in circulating blood. It forms urokinase from prourokinase. It lyses the plaminogen activator inhibitor and enhances the concentration of plasminogen activator of tissues.  Under normal metabolic conditions, equilibrium is set between blood clotting and degradation of blood fibrin. This equilibrium may be disturbed due to tissue damage, gene modification or disorders like diabetes.

 

Under these conditions if Nattokinase is administered into the human body the normal blood circulation is retained[1-3].  Nattokinase is also used in the treatment of hypertension, cerebral hemorrhage, aggregation of platelets and high blood viscosity. Researchers had exploited various microbes to produce Nattokinase. The microbes which are able to produce Nattokinase include Streptococcus pyogenes, Aeromonas hydrophila, Serratia E15, Bacillus natto, Bacillus amyloliquefaciens, Fusarium oxysporum and Mucor species[4].

 

Various genomic and Protein databases are available online from which users can freely retrieve the desired organism’s gene and protein sequences. Computational tools are exploited to model and validate protein structures based on corresponding protein sequences or genomic sequences. Protein modeling is a developing area. Scientists from various fields like mathematics, physical sciences and biological sciences had combined their ideas to develop new software tools (Bioinformatics tools) to predict and improve protein models. In recent years many web-based bioinformatics tools are developed which are user friendly and to use them user does not require any programming knowledge[5].

 

In the present study, the amino acid sequence (FASTA format) of Nattokinase enzyme was obtained from UniProt server and submitted to Phyre2 tool to generate 3-D model of Nattokinase. The quality of modeled structure of Nattokinase was verified and validated using Verify3D and ProSA software tools.

 

2. MATERIALS AND METHODS:

2.1. Collection of amino acid sequence of Nattokinase enzyme:

The sequence of Nattokinase enzyme was obtained from UniProt server which is a data collection centre of various proteins of living organisms available in different protein databanks[6].

 

2.2. Construction of 3-D model of Nattokinase enzyme:

The Nattokinase enzyme sequence (query sequence) was entered in Phyre2 server for generation of its model structure based on its template. In Phery2 the protein sequence which shows high degree of similarity with query sequence is regarded as template, based on which the model of the query protein is generated[7].

 

2.3. Verification of Nattokinase enzyme model: The quality of  structure model of Nattokinase enzyme was checked in Verify3D and ProSA programs. Verify3D and ProSA are the mostly widely used reliable programs for the validation of protein models in which the model quality is validated based on overall score generated in connection with the features of amino acids present in a protein sequence [8, 9].

 

3. RESULTS AND DISCUSSION:

3.1. Nattokinase enzyme sequence:

The Nattokinase enzyme sequence of Bacillus subtilis sub sp. natto was obtained in FASTA format from UniProt server.  The FASTA format sequence of Nattokinase is depicted below.

MRSKKLWISLLFALTLIFTMAFSNMSAQAAGKSSTEKKYIVGFKQTMSAMSSAKKKDVISEKGGKVQKQFKYVNAAAATLDEKAVKELKKDPSVAYVEEDHIAHEYAQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVAPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSSNQRASFSSVGSELDVMAPGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ

 

The UniProt server is a Universal Protein Knowledgebase which is formed by the combination of TreEMBL, PIR protein and SwissProt databases. From Uniprot users can easily download the desired protein sequences freely[10].

 

3.2. Generation of Nattokinase enzyme 3-D model:

The FASTA format of Nattokinase enzyme sequence (query sequence) was submitted to Phyre2 server. In Phyre2 server the Nattokinase enzyme sequence aligns with different similar protein sequences available in the Phyre2 databank and the protein with highest similarity was selected as template to build the Nattokinase model. The best matching template determined in Phyre2 was c3whiA. The alignment of sequences of c3whiA and Nattokinase was shown in figure-1. Based on the template, c3whiA model (Figure-2) the structure model of Nattokinase enzyme (Figure-3) was built.  Phyre2 is online web-based program which is used to construct three-dimensional models of proteins. The amino acid sequence of a given protein is needed to be submitted to Phyre2 to develop a corresponding protein model[11].

 

Figure 1: Alignment of Nattokinase enzyme (query sequence) and its template (c3whiA) in Phyre2


 

Figure 2: Template (c3whiA) 3-D model

 

Figure 3: Nattokinase enzyme 3-D model

 

3.3. Validation of modeled structure of Nattokinase enzyme:

The model structure of Nattokinase developed in Phyre2 was validated using the most widely used model quality determining programs, Verify3D and ProSA. Both Verify3D and ProSA programs verify the consonance between given protein model and its amino acid sequence.  The PDB format of a protein is submitted as input in Verify3D and ProSA programs. Then in these programs a protein model quality score is generated based on congruency of various properties of amino acids present in protein sequence with that of three-dimensional protein model. In Verify3D the average 3D-1D score of 95.39% amino acids of Nattokinase enzyme was >=0.2 (Figure-4) inferring its good model quality.  To confirm the validity of a protein model at least 80% of its amino acids average 3D-1D score must be >=0.2 in Verify3D program. The overall Z-score (Figure-5) for Nattokinase enzyme generated in ProSA program was -9.74 indicating the reliability of the Nattokinase enzyme model developed in Phyre2. The score generated for Nattokinase model in ProSA correlates to the previously calculated scores of predetermined structures of proteins in protein databank [12, 13].


 

Figure4: Generation of 3D-1D score based on Amino acids organization in Verify3D program

(Blue colour: Averaged score and Green Colour: Raw score)


 

Figure5: The Z-score (represented by black dot) of Nattokinase enzyme generated by NMR (dark blue) and X-ray (light blue) in ProSA  depending on the features of amino acids present in sequence.

 

4. CONCLUSION:

In the present work the Bioinformatics software tools were exploited to study the Nattokinase enzyme structural details. Nattokinase enzyme sequence obtained in UniProt was used to develop and validate the structure model of Nattokinase enzyme. The microbial Nattokinase enzyme has multiple therapeutic applications in addition to its primary anti-clotting usage. Such Nattokinase can be commercially exploited. Further the Nattokinase activity can be improved using bioinformatics tools. The amino acids of Nattokinase enzyme at certain specific sites can be altered by employing computational biology tools like Discovery Studio and same improved protein can be produced by microbes with modifications in the corresponding sites of  Nattokinase gene.

 

5. CONFLICT OF INTEREST:

There is no conflict of interest.

 

6. REFERENCES:

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Received on 15.10.2019            Modified on 18.12.2019

Accepted on 29.01.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(9):4135-4138.

DOI: 10.5958/0974-360X.2020.00730.1