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==E.Coli Gyrase== | |||
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{{STRUCTURE_1ab4| PDB=1ab4 | SCENE=56/568026/Coloured/1}} | {{STRUCTURE_1ab4| PDB=1ab4 | CAPTION=A-subunit (monomer), resolution 2.80Å, 1ab4.pdb| SCENE=56/568026/Coloured/1}} | ||
[[Image:1ab4.png|left|200px]] | [[Image:1ab4.png|left|200px]] | ||
=='''Introduction | ==='''Introduction'''=== | ||
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---- | ---- | ||
=='''Structure'''== | |||
==='''Structure'''=== | |||
[[Image:Gyrasegene.jpg]] | [[Image:Gyrasegene.jpg]] | ||
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There is a '''<scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain''', folowing the tower, with a helical core at the tail region. Two long helices ( | There is a '''<scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain''', folowing the tower, with a helical core at the tail region. Two long helices ( | ||
<scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix ( | <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is '''involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene>.''' | ||
<scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is '''involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene>.''' | |||
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The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. | The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. | ||
GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. | '''GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates'''. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. | ||
The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA. | The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA. | ||
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''' | ==='''Mechanism'''=== | ||
40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA. | 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA. | ||
N- and C-gates that open or close to allow T-segment transport through both | N- and C-gates that open or close to allow T-segment transport through both the protein and the cleaved G-segment. | ||
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=='''Reaction'''== | ==='''Reaction'''=== | ||
The process of DNA supercoiling by gyrase is the following : | The process of DNA supercoiling by gyrase is the following : | ||
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# Translocation | # Translocation | ||
The A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity. | The '''A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.''' | ||
The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)so that the linking number (Lk) changes in step of 2. The ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA. | '''The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)''' so that the linking number (Lk) changes in step of 2. The '''ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA.''' | ||
The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase. | '''The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.''' | ||
The DNA supercoiling reaction requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>, and is stimulated in the presence of spermidine. | The DNA supercoiling reaction '''requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>''', and is stimulated in the presence of spermidine. | ||
Therefore, the gyrase could also catalyse the following reaction : | Therefore, the gyrase could also catalyse the following reaction : | ||
* Relaxation of negative/positive supercoils | * Relaxation of negative/positive supercoils | ||
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* Unknotting DNA strands | * Unknotting DNA strands | ||
Contrary to the introduction of negative supercoils, some of these reactions don't necessarily need the energy from ATP-hydrolysis. | Contrary to the introduction of negative supercoils, '''some of these reactions don't necessarily need the energy from ATP-hydrolysis.''' | ||
The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang. | The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang. | ||
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The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA. | The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA. | ||
The gyrase could be inhibited by both the quinolones and | The gyrase could be inhibited by both the [http://en.wikipedia.org/wiki/Quinolone quinolones] and [http://en.wikipedia.org/wiki/Coumarin coumarins]. | ||
Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP, so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known. | |||
'''Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP''', so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known. | |||
However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino | |||
'''Quinolones''' inhibit DNA supercoiling by gyrase, it has even the ability to '''induce DNA cleavage''' instead. It appears that '''these drugs will arrest any gyrase rection involving double-strand DNA breakage.''' | |||
However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acids 67 and 106. | |||
There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase. | There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase. | ||
==See Also== | ===See Also=== | ||
*[[Gyrase|Gyrase]] | *[[Gyrase|Gyrase]] | ||
*[[Topoisomerase|Topoisomerase]] | *[[Topoisomerase|Topoisomerase]] | ||
== | ===References=== | ||
<ref group="xtra">PMID:009278055</ref><references group="xtra"/> | <ref group="xtra">PMID:009278055</ref><references group="xtra"/> | ||
[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
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[[Category: Topoisomerase ii]] | [[Category: Topoisomerase ii]] | ||
*Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375. | *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375. | ||
*Nicole M. Baker, Steven Weigand, Sarah Maar-Mathiasand Alfonso Mondrago´n "Solution structures of DNA-bound gyrase" Nucleic Acids Res. 2011 January; 39(2): 755–766. | |||
