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Academic Sciences International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 5, Suppl 4, 2013 Research Article INSILICO ANALYSIS OF GYRASE SUBUNITS A AND B IN PROKARYOTES SUMEET R. DESHMUKH, PRITEE CHUNARKAR AND MD ATAUL ISLAM* Department of Bioinformatics, Rajiv Gandhi Institute of IT and Biotechnology, Bharati Vidyapeeth Deemed University, Pune-Satara Road, Katraj, Pune 411046. Email: [email protected] Received: 03 Sep 2013, Revised and Accepted: 05 Oct 2013 ABSTRACT Objective: The present study focused on type II topoisomerases, especially Gyrase and tried to investigate the evolutionary aspect by studying the phylogeny due to the wealth of information available on these enzymes. Method: The sequences were retrieved from Uniprot, aligned using ClustalW and phylogenetic analysis was carried out to predict the structure function relationship amongst the DNA gyrases. Result: Many conserved regions including AAMRYTE are found in A subunit and ATPase activity is associated with B subunit, suggesting the conserved evolutionary trend. Conclusion: The DNA breakage and reunion by subunit A is assisted by ATPase activity shown by subunit B of Gyrases in prokaryotes reveals the importance of the two genes being conserved through the evolutionary period. Keywords: Gyrase, Topology, Phylogenetic analysis, Evolution. INTRODUCTION DNA performs two functions and manipulations. All these processes such as supercoiling-relaxation, catenationdecatenation and knotting-unknotting (folding-unfolding) of DNA are done with the help of DNA topoisomerases. Key cellular processes such as replication, transcription, recombination and chromosome segregation require topological events. Thus, the enzymes are indispensable for the cell survival, and hence are ubiquitous. The topoisomerases are classified into two distinct subclasses based on the mechanism of the reaction. The type I topoisomerases break one strand of DNA and pass the other stand through the nick created and change the linking number in one step. On the other hand, type II enzymes cleave both strands of DNA and pass the duplex through the ‘DNA gate’ resulting in the change of linking number in steps of two [1]. DNA topoisomerases modulate DNA structure by inter-converting different DNA topoisomers [2]. Gyrases are bacterial type II topoisomerases that use the chemical energy of ATP hydrolysis to introduce negative supercoils into DNA [3]. The active form of gyrase is a heterotetramer formed by two GyrA and two GyrB subunits [4]. Comparison of the primary sequence suggests that eukaryotic topoisomerase II is evolved by the fusion of the GyrA and GyrB which are the genes of DNA gyrase, the eubacterial possesses the same function as that of topoisomerase II but performs functions in different areas (counterparts) [5]. In this compilation, we have focused our attention on type II topoisomerases, especially Gyrase and tried to investigate the evolutionary aspect by studying the phylogeny. This is due to the wealth of information available on these enzymes, their indispensability and the degree of conservation amongst the genes from variety of organisms. MATERIALS AND METHODS GyrB and GyrA polypeptide sequences have been characterized from several bacteria. The GyrB and GyrA polypeptide sequences are retrieved from Uniprot database. Tables 1 and 2 summarize the source and the length of the derived polypeptides. Table1: Polypeptide sequences of GyrA, sequence was retrieved from Uniprot Organism Aeromonas salmonicida Bacillus halodurans Brachyspira hyodysenteriae (strain ATCC 49526 / WA1) Campylobacter fetus Chlamydia pneumonia Clostridium acetobutylicum Escherichia coli (strain K12) Erwinia carotovora Length 922 AA 833 AA 834 AA 862 AA 834 AA 830 AA 874 AA 878 AA Accession number P48369 O50628 C0R046 P47235 Q9Z8R4 P94605 P0AES4 P41513 Reference [6] [7] [8] [9] [10] [11] [12] [13] Table 2: Polypeptide sequences of GyrB, sequence was retrieved from Uniprot Organism Haloferax volcanii (strain ATCC 29605 / DSM 3757 / JCM 8879 / NBRC 14742 / NCIMB 2012 / VKM B-1768 / DS2) Haloarcula marismortui (strain ATCC 43049 / DSM 3752 / JCM 8966 / VKM B-1809) Haloterrigena turkmenica (strain ATCC 51198 / DSM 5511 / NCIMB 13204 / VKM B-1734) Halalkalicoccus jeotgali (strain DSM 18796 / CECT 7217 / JCM 14584 / KCTC 4019 / B3) Vibrio cholera Gordonia neofelifaecis Prevotella timonensis Brucella pinnipedialis Escherichia coli Filifactor alocis (strain ATCC 35896 / D40 B5) Length 639 AA Accession number D4GZ01 Reference [15] 642 AA. 644 AA 635 AA 805 AA. 634 AA 657 AA 813 AA 804 AA 638 AA Q5V4R6 D2RUH9 D8J639 A3GYE1 F1YLA3 D1VZA2 C9TSV3 D6IFU3 D6GSU7 [16] [17] [18] [19] [20] [21] [22] [23] [24] Islam et al. Int J Pharm Pharm Sci, Vol 5, Suppl 4, 339-345 Clustal X was used for multiple sequence alignment of GyrA and GyrB [25]. The multiply aligned sequences were subjected to PHYLIP analysis [1]. The output was then analysed by applying neighborjoining method. Dendograms were generated. Superfamily 1.75 HMM library and genome assignment was used for finding out the domains and there functions with schematic representation. RESULTS AND DISCUSSION This compilation and alignment of GyraseA and GyraseB is an attempt to compile complete sequences and determine the extent of phylogenetic relationships. These reports show conservation of amino acid sequence in gyrase. Hence, we have presented the alignment of all deduced polypeptide sequences of GyraseA and GyraseB in Figs. 1 and 2. In order to avoid errors in alignment and phylogeny analyses, we have omitted partial sequences. We found the DNA breakage-reunion site of subunit A has the sequence AAMRYTE common to all the members which also being presented [1]. Beside this we also found MSVIV, RALPD, GNFGSID, GPDFPT common to all the members still function of this sequence is unknown and the residue Tyrosine mostly present on 122 nd position of GyraseA covalently attached to DNA through Phosophodiester bond, which indicates its important role in the functioning of gyrase A to hold DNA after breaking the DNA strands in order to again rejoin in it [26]. Fig. 1: Multiple Sequence alignment of Gyrase subunit A using ClustalX 340 Islam et al. Int J Pharm Pharm Sci, Vol 5, Suppl 4, 339-345 Fig. 2: Multiple Sequence alignment of Gyrase subunit B using ClustalX The subunit B of bacterial type II topoisomerases shows identical patches of amino acids scattered throughout the sequences. Retaining ATPase activity a characteristic of all type II topoisomerases. The N-terminal 43 kDa fragment of E. coli GyrB is known to retain ATPase activity. The crystal structure of this domain complexed with ADPNP has revealed the direct interaction between the protein and the cofactor [27]. The sequence M-Y-H-I-T is conserved in all the sequence from the position 40-42-43-44-45 respectively. Besides this patches of amino acid conserved all over the sequences were observed. The subunits of bacterial type II topoisomerases were further analyzed to understand the evolutionary relatedness. The rooted trees are shown in Figs. 3 and 4. In Fig. 3, three clusters can be observed based on evolutionary time. Subunit A of Campylobacter sp is the out-group for subunit A of rest of the organisms. A subunits of Escherichia, Erwinia, Chlamydia and Aeromonas are closely related to each other forming a sister group in which Chlamydia is the out-group for rest three where as subunits from Bacillus, Haemophilus, Brachyspira and Clostridium share the other group. 341 Islam et al. Int J Pharm Pharm Sci, Vol 5, Suppl 4, 339-345 AERSA ECOLI ERWCA CHLPN BACHD HAEIN BRAHW CLOAB CAMFE Fig. 3: Evolutionary relationship among Prokaryotes of GyraseA mentioned in Table 1 HALVD HALTV HALJB HALMA 9BACT FILAD 9ACTO VIBCH ECOLX 9RHIZ Fig. 4: Evolutionary relationship among Prokaryotes of GyraseB mentioned in Table 2 In Fig. 4, Subunit B of Brucella sp is the out-group for all the members. Vibrio and Escherichia share the sister group for subunit B. For the third sister group, subunit B of Gordonia is the out-group where as further two subgroups are generated namely, Haloarcula, Prevotella and Filifactor in one whereas Haloferax, Haloterrigena and Halalkalicoccus share the other subgroup. The genes for subunit A and B both showed the evolution on time scale. Domains of GyraseA and GyraseB with their function are determined with the help of superfamily tool available on Expasy server. Schematic representation has being shown in Figs. 5 and 6 respectively and functions of the other domains are listed in Tables 3, 4, 5 and 6. Table 3: Domains in GyraseA subunit Accession no. P48369 O50628 C0R046 P47235 Q9Z8R4 P94605 P0AES4 P41513 P43700 Domain no. 1 2 3 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 Region 30-522 534-717 758-896 31-486 498-819 49-507 520-832 35-489 501-661,695-851 31-487 500-819 29-485 497-817 30-521 533-851 30-521 533-848 31-518 Family Type II DNA topoisomerase GyrA/ParC C-terminal domain-like GyrA/ParC C-terminal domain-like Type II DNA topoisomerase GyrA/ParC C-terminal domain-like Type II DNA topoisomerase GyrA/ParC C-terminal domain-like Type II DNA topoisomerase GyrA/ParC C-terminal domain-like Type II DNA topoisomerase GyrA/ParC C-terminal domain-like Type II DNA topoisomerase GyrA/ParC C-terminal domain-like Type II DNA topoisomerase GyrA/ParC C-terminal domain-like Type II DNA topoisomerase GyrA/ParC C-terminal domain-like Type II DNA topoisomerase 342 Islam et al. Int J Pharm Pharm Sci, Vol 5, Suppl 4, 339-345 Fig. 5: Domains are showed by Different colors of GyraseA. by using superfamily Fig. 6: Domains are showed by Different colors of GyraseB by using superfamily 343 Islam et al. Int J Pharm Pharm Sci, Vol 5, Suppl 4, 339-345 Table 4: Domains in Gyrase subunit B Accession no. D4GZ01 Q5V4R6 D2RUH9 D8J639 A3GYE1 F1YLA3 D1VZA2 C9TSV3 D6IFU3 D6GSU7 Domain no. 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Region 399-633 7-222 225-396 402-636 8-222 225-399 404-638 7-215 231-401 397-629 7-221 224-394 395-561,731-796 4-213 221-393 437-678 20-223 263-434 407-650 11-216 230-403 405-574,739-805 15-221 233-403 394-560,730-795 4-220 221-392 398-630 10-218 227-395 Family Type II DNA topoisomerase DNA gyrase/MutL, N-terminal domain DNA gyrase/MutL, second domain Type II DNA topoisomerase DNA gyrase/MutL, N-terminal domain DNA gyrase/MutL, second domain Type II DNA topoisomerase DNA gyrase/MutL, N-terminal domain DNA gyrase/MutL, second domain Type II DNA topoisomerase DNA gyrase/MutL, N-terminal domain DNA gyrase/MutL, second domain Type II DNA topoisomerase DNA gyrase/MutL, N-terminal domain DNA gyrase/MutL, second domain Type II DNA topoisomerase DNA gyrase/MutL, N-terminal domain DNA gyrase/MutL, second domain Type II DNA topoisomerase DNA gyrase/MutL, N-terminal domain DNA gyrase/MutL, second domain Type II DNA topoisomerase DNA gyrase/MutL, N-terminal domain DNA gyrase/MutL, second domain Type II DNA topoisomerase DNA gyrase/MutL, N-terminal domain DNA gyrase/MutL, second domain Type II DNA topoisomerase DNA gyrase/MutL, N-terminal domain DNA gyrase/MutL, second domain Table 5: Molecular functions of domains of Gyrase subunit A mentioned in Fig. 5 Accession number CORO46 O50628 POAES4 P41513 P43700 P47235 P48369 P94605 Q928R4 Molecular function ATP DNA Topoisomerase Activity (ATP binding hydrolysis) Seq. specific DNA binding transcriptase factor activity Table 6: Molecular functions of domains of Gyrase subunit B mentioned in Fig. 6 Accession number A3GYE1 C9TSV3 D1V2A2 D2RUH9 D4GZO1 D6GSU7 D61FU3 D8J639 F1YLA3 Q5V4R6 Molecular function ATP binding DNA Topoisomerase Activity (ATP hydrolysis) GyraseB have an important role as ATPase which is required for Cutting and rejoining DNA strand, as topoisomerase II enzyme. Topoisomerase ll enzyme consists of two subunits, A and B, and the active enzyme is an A2B2 tetrameric complex [28]. CONCLUSION The DNA breakage and reunion by subunit A is assisted by ATPase activity shown by subunit B of Gyrases in prokaryotes reveals the importance of the two genes being conserved through the evolutionary period. This investigation may lead to the proper understanding of the topological events in the prokaryotic cell, in order to carry out distant experimentation requiring such manipulations. REFERENCES 1. 2. Madhusudhan K, Nagaraja V. Alignment and phylogenetic analysis of type II DNA topoisomerases. J. Biosci. 1996; 21: 613619. Champoux JJ. DNA topoisomerases: Structure, function, and mechanism. Annu. Rev. Biochem. 2001; 70: 369–413. 344 Islam et al. Int J Pharm Pharm Sci, Vol 5, Suppl 4, 339-345 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Gellert M, Mizuuchi K, O’Dea MH, Nash H. DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc. Natl. 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