Sandbox Reserved 1499

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===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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The oxygen from Ser40 binds to the carbon from the peptide bond between the two D-alanines, which causes the formation of the acylenzyme complex after the terminal D-alanine leaves.
The oxygen from Ser40 binds to the carbon from the peptide bond between the two D-alanines, which causes the formation of the acylenzyme complex after the terminal D-alanine leaves.
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===Binding site===
===Binding site===
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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.
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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.
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Ser 83, specifically, is thought to be capable of stabilising the leaving group during the acylation reaction through hydrogen bonds.
Ser 83, specifically, is thought to be capable of stabilising the leaving group during the acylation reaction through hydrogen bonds.
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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 stabilise the carbonyl oxygen from the D-Ala-D-Ala peptide bond.
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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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The Ser106, Thr210, Thr212 and Arg244 residues stabilise the C-terminal of the peptide through hydrogen bonds and bonds through water molecules (see structure [[3itb]]).
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It can be seen that PBP6 presents both a binding site and an active site with a precise configuration that makes it specific to its substrate.
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The Ser106, Thr210, Thr212 and Arg244 residues stabilise the C-terminal of the substrate thanks to hydrogen bonding and bonds through water molecules (see structure [[3itb]]).
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These features show that PBP6 presents both a binding site and an active site with a precise configuration that makes it highly specific to its substrate.
===Ampicillin mimicry===
===Ampicillin mimicry===
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 enzyme substrate which allow it to bind to its active site. It however also possesses characteristics which allow it to stop the enzyme from functionning. It can use the same mechanisms as the enzyme substrate <scene name='80/802673/Active_site_amp/2'>to insert into the active site</scene>, and specifically the oxyanion hole.
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Thanks to its O3 carboxylate group for example, it can insert It also forms a hydrogen bond with the Thr212 side chain.
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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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Similarly, Arg244 also binds to the C3 carboxylate group through a water molecule.
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However, PBP6 cannot hydrolyse the amide bond from ampicillin. This is due to the bond in question being contained inside the beta-lactam cycle of the antibiotic. Additionally, the sulfur atom from the ampicillin molecule sterically hinders the interaction between Lys43 and the
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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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Similarly, the C3 carboxylate group can bind to Arg244 through a water molecule, as the substrate would.
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However, PBP6 cannot hydrolyse the amide bond from ampicillin. This is due to the bond in question being contained inside the beta-lactam cycle of the antibiotic. Additionally, the sulfur atom from the ampicillin molecule hinders the interaction between Lys43 and the
<scene name='80/802673/Cat_water/2'>surrounding water molecules</scene>. As a result, no water molecule can be protonated in order to perform the hydrolysis.
<scene name='80/802673/Cat_water/2'>surrounding water molecules</scene>. As a result, no water molecule can be protonated in order to perform the hydrolysis.
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It can be seen that through its structure, ampicilline imitates the natural substrate of PBP6 and stops it from performing its catalysis.
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Through its pentapeptidic-imitating structure, ampicilline mimics the natural substrate of PBP6 and stops it from performing its catalysis.
</StructureSection>
</StructureSection>
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== Relevance==
== Relevance==
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The resolution of this structure played an important role in understanding the method of action of beta-lactam antibiotics<ref>DOI 10.1016/j.mib.2010.09.008</ref>. Due to the increase of antibiotic-resistant bacteria, 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>. Due to the increase of antibiotic-resistant bacteria, it is important to understand the method of action of antibiotics in their every detail, so that treatments for resistent bacteria can be devised.
== References ==
== References ==
<references/>
<references/>

Revision as of 22:12, 11 January 2019

This Sandbox is Reserved from 06/12/2018, through 30/06/2019 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1480 through Sandbox Reserved 1543.
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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 functionning of the active site of PBP6 and of the role of pentapeptidic imitation by ampicillin. [2]

Contents


Function

As a DD-carboxypeptidase, the function of PBP6 is to participate in the transpeptidation which occurs during the biosynthesis of peptidoglycan. 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 terminal D-alanines of the N-acetylmuramic acid. 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[3]. Due to the increase of antibiotic-resistant bacteria, it is important to understand the method of action of antibiotics in their every detail, so that treatments for resistent 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. ↑ 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
  3. ↑ 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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