Download INSILICO ANALYSIS OF GYRASE SUBUNITS A AND B IN PROKARYOTES

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
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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.
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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
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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
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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.
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