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{{Sandbox_Reserved_ESBS}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE -->
{{Sandbox_Reserved_ESBS}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE -->
==Penicillin-binding protein 6 from ''Escherichia coli''==
==Penicillin-binding protein 6 from ''Escherichia coli''==
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The Penicillin-binding protein 6 (PBP6) from ''Escherichia coli'' is a DD-carboxypeptidase which plays an important role in the creation of the bacterial cell wall. It belongs to the group of PBP of low molecular mass. Its structure was determined by Chen et al.<ref>PMID:19807181</ref>. These results allow for the study of the functionning of the active site of PBP6 and of the role of pentapeptidic imitation by ampicillin.
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The Penicillin-binding protein 6 (PBP6) from ''Escherichia coli'' is a DD-carboxypeptidase which plays an important role in the creation of the bacterial cell wall. It belongs to the group of PBP of low molecular mass. Its structure was determined by Chen et al.<ref>PMID:19807181</ref>. These results allow for the study of the functioning of the active site of PBP6 and of the role of pentapeptidic imitation by ampicillin.
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== Function ==
== Function ==
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As a DD-carboxypeptidase, the function of PBP6 is to participate in the transpeptidation which occurs during the biosynthesis of peptidoglycan<ref>DOI 10.1016/j.sbi.2010.09.014</ref>. More specifically, it cleaves the peptide bond between the two terminal D-alanines of the pentapeptidic muramyl peptides of sequence ''L''-Ala-''D''-Glu-m-A2pm-''D''-Ala-''D''-Ala. This then allows transpeptidases to create peptidoglycan cross-links which stabilise the cell wall.
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As a DD-carboxypeptidase, the function of PBP6 is to participate in the transpeptidation which occurs during the biosynthesis of peptidoglycan <ref>PMID:18266856</ref>. More specifically, it cleaves the peptide bond between the two terminal D-alanines of the pentapeptidic muramyl peptides of sequence ''L''-Ala-''D''-Glu-m-A2pm-''D''-Ala-''D''-Ala. This then allows transpeptidases to create peptidoglycan cross-links which stabilise the cell wall.
The cleavage reaction takes place in two steps.
The cleavage reaction takes place in two steps.
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#Firstly, the PBP6 binds to carbonyl group in the peptide bond between the two terminal D-alanines of the N-acetylmuramic acid. This forms a high-energy tetrahedric intermediate called the acylenzyme.
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#Firstly, the PBP6 binds to carbonyl group in the peptide bond between the two C-terminal D-alanines of the N-acetylmuramic acid<ref>DOI 10.1016/j.sbi.2010.09.014</ref>. This forms a high-energy tetrahedric intermediate called the acylenzyme.
#The acylenzyme allows the medium to reach the carbonyle group. As a result, a water molecule can attack the group, causing the cleavage of the tetrahedral structure.
#The acylenzyme allows the medium to reach the carbonyle group. As a result, a water molecule can attack the group, causing the cleavage of the tetrahedral structure.
== Structure==
== Structure==
<StructureSection load='3ita' size='340' side='right' caption='PBP6, [[Resolution|resolution]] 1.80&Aring;' scene='80/802673/Default/1'>
<StructureSection load='3ita' size='340' side='right' caption='PBP6, [[Resolution|resolution]] 1.80&Aring;' scene='80/802673/Default/1'>
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PBP6 is a monomeric enzyme with an active site near its N-terminal domain.
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PBP6 is a monomeric enzyme with a C-terminal domain rich in beta-sheets and an active site localised near its N-terminal domain<ref>PMID:22126997</ref>.
===Active site===
===Active site===
The amino acids that compose the <scene name='80/802673/Active_site_1/1'>active site</scene> are: Ser40 and Lys43, which are organised in a catalytic diad through a hydrogen bond, alongside with Ser106, Asp108, Lys209 and Thr210.
The amino acids that compose the <scene name='80/802673/Active_site_1/1'>active site</scene> are: Ser40 and Lys43, which are organised in a catalytic diad through a hydrogen bond, alongside with Ser106, Asp108, Lys209 and Thr210.
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Other amino acids allow the selective fixation of the ligand to the enzyme. The residues 79 to 83, 212 to 218 and 242 to 248 are responsible for this. These residues undergo a conformation change during substrate fixation which changes the configuration of the entire enzyme and gives the substrate access to the active site.
Other amino acids allow the selective fixation of the ligand to the enzyme. The residues 79 to 83, 212 to 218 and 242 to 248 are responsible for this. These residues undergo a conformation change during substrate fixation which changes the configuration of the entire enzyme and gives the substrate access to the active site.
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Ser 83, specifically, is thought to be capable of stabilising the leaving group during the acylation reaction through hydrogen bonds.
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Ser83, specifically, is thought to be capable of stabilising the leaving group during the acylation reaction through hydrogen bonds.
Another important part of the selective fixation process is the oxyanion hole, which is a hole in the enzyme structure created by the backbone nitrogens of Ser40 and Thr212. These nitrogens can interact with the carbonyl oxygen from the D-Ala-D-Ala peptide bond, thus stabilising it.
Another important part of the selective fixation process is the oxyanion hole, which is a hole in the enzyme structure created by the backbone nitrogens of Ser40 and Thr212. These nitrogens can interact with the carbonyl oxygen from the D-Ala-D-Ala peptide bond, thus stabilising it.
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Ampicillin stops the synthesis of peptidoglycan by competitive inhibition of PBP6. [[3ita]] shows the acylenzyme complex of PBP6 with ampicillin.
Ampicillin stops the synthesis of peptidoglycan by competitive inhibition of PBP6. [[3ita]] shows the acylenzyme complex of PBP6 with ampicillin.
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Ampicillin presents structural characteristics which are similar to the PBP6's substrate. These characteristics allow it to bind to the enzyme and access its active site. It however also possesses characteristics which allow it to stop the enzyme from functionning.
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Ampicillin presents structural characteristics which are similar to the PBP6's substrate. These characteristics allow it to bind to the enzyme and access its active site. It however also possesses characteristics which allow it to stop the enzyme from functioning.
It uses the same mechanisms as the enzyme substrate <scene name='80/802673/Active_site_amp/2'>to insert itself into the active site</scene>. Thanks to its O3 carboxylate group for example, it can mimic the carbonyl oxygen from the peptide bond and make use of the oxyanion hole described previously. The oxygen atom in question additionally forms a hydrogen bond with the Thr212 side chain.
It uses the same mechanisms as the enzyme substrate <scene name='80/802673/Active_site_amp/2'>to insert itself into the active site</scene>. Thanks to its O3 carboxylate group for example, it can mimic the carbonyl oxygen from the peptide bond and make use of the oxyanion hole described previously. The oxygen atom in question additionally forms a hydrogen bond with the Thr212 side chain.
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== Relevance==
== Relevance==
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The resolution of this structure plays an important role in understanding the exact method of action of beta-lactam antibiotics<ref>DOI 10.1016/j.mib.2010.09.008</ref>. Since antibiotic resistance is often based on small changes in protein conformation at the molecular level, it is important to understand the method of action of antibiotics in their every detail, so that treatments for resistent bacteria can be devised.
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The resolution of this structure plays an important role in understanding the exact method of action of beta-lactam antibiotics<ref>DOI 10.1016/j.mib.2010.09.008</ref>. Since antibiotic resistance is often based on small changes in protein conformation at the molecular level, it is important to understand the method of action of antibiotics in their every detail, so that treatments for resistant bacteria can be devised.
== References ==
== References ==
<references/>
<references/>

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Penicillin-binding protein 6 from Escherichia coli

The Penicillin-binding protein 6 (PBP6) from Escherichia coli is a DD-carboxypeptidase which plays an important role in the creation of the bacterial cell wall. It belongs to the group of PBP of low molecular mass. Its structure was determined by Chen et al.[1]. These results allow for the study of the functioning of the active site of PBP6 and of the role of pentapeptidic imitation by ampicillin.

Contents


Function

As a DD-carboxypeptidase, the function of PBP6 is to participate in the transpeptidation which occurs during the biosynthesis of peptidoglycan [2]. More specifically, it cleaves the peptide bond between the two terminal D-alanines of the pentapeptidic muramyl peptides of sequence L-Ala-D-Glu-m-A2pm-D-Ala-D-Ala. This then allows transpeptidases to create peptidoglycan cross-links which stabilise the cell wall.

The cleavage reaction takes place in two steps.

  1. Firstly, the PBP6 binds to carbonyl group in the peptide bond between the two C-terminal D-alanines of the N-acetylmuramic acid[3]. This forms a high-energy tetrahedric intermediate called the acylenzyme.
  2. The acylenzyme allows the medium to reach the carbonyle group. As a result, a water molecule can attack the group, causing the cleavage of the tetrahedral structure.

Structure

PBP6, resolution 1.80Å

Drag the structure with the mouse to rotate

Relevance

The resolution of this structure plays an important role in understanding the exact method of action of beta-lactam antibiotics[5]. Since antibiotic resistance is often based on small changes in protein conformation at the molecular level, it is important to understand the method of action of antibiotics in their every detail, so that treatments for resistant bacteria can be devised.

References

  1. ↑ Chen Y, Zhang W, Shi Q, Hesek D, Lee M, Mobashery S, Shoichet BK. Crystal structures of penicillin-binding protein 6 from Escherichia coli. J Am Chem Soc. 2009 Oct 14;131(40):14345-54. PMID:19807181 doi:10.1021/ja903773f
  2. ↑ Sauvage E, Kerff F, Terrak M, Ayala JA, Charlier P. The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev. 2008 Mar;32(2):234-58. doi: 10.1111/j.1574-6976.2008.00105.x., Epub 2008 Feb 11. PMID:18266856 doi:http://dx.doi.org/10.1111/j.1574-6976.2008.00105.x
  3. ↑ Mattei PJ, Neves D, Dessen A. Bridging cell wall biosynthesis and bacterial morphogenesis. Curr Opin Struct Biol. 2010 Dec;20(6):749-55. doi: 10.1016/j.sbi.2010.09.014., Epub 2010 Oct 26. PMID:21030247 doi:http://dx.doi.org/10.1016/j.sbi.2010.09.014
  4. ↑ van Heijenoort J. Peptidoglycan hydrolases of Escherichia coli. Microbiol Mol Biol Rev. 2011 Dec;75(4):636-63. doi: 10.1128/MMBR.00022-11. PMID:22126997 doi:http://dx.doi.org/10.1128/MMBR.00022-11
  5. ↑ Llarrull LI, Testero SA, Fisher JF, Mobashery S. The future of the beta-lactams. Curr Opin Microbiol. 2010 Oct;13(5):551-7. doi: 10.1016/j.mib.2010.09.008. Epub, 2010 Sep 29. PMID:20888287 doi:http://dx.doi.org/10.1016/j.mib.2010.09.008
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