Hen Egg-White (HEW) Lysozyme

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<StructureSection load='1hew' size='450' side='right' scene='' caption='Glycosylated hen egg white lysozyme (PDB code [[1hew]])'>
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__TOC__
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[[Image:1hewB.gif|frame|Hen egg-white lysozyme, 1HEW (scroll down to the [[#Structure_of_Lysozyme|structure section]] for interactive figure)]]
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'''Lysozyme''' - also known as muramidase - is a powerful enzyme found in abundance in tears, saliva, and human milk. In humans, it is encoded in the ''LYZ'' gene. Since it is a small, easily available, and highly stable protein, it has been subject to extensive research regarding its function and structure.
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==Introduction==
==Introduction==
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Lysozyme acts as a non-specific defense against bacteria and fungi. It is a component of the innate immune system, and is an important part of an infant's diet to ward off diarrhea. It is an enzyme known for its ability to degrade the polysaccharide architecture of many kinds of cell walls, normally for the purpose of protection against bacterial infection<ref>PMID 28934357</ref>. The structure of hen egg white (HEW) lysozyme, the focus of this article, is shown on the right. The antibacterial activity of hen egg white was first described by Laschtschenko in 1909<ref>Laschtschenko,P. (1909)
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Über die keimtötende und entwicklungshemmende Wirkung von Hühnereiweiss. Z. Hyg. Infektionskrankh.,64,419-427.
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</ref>. It was characterized and named “lysozyme” by Alexander Fleming, the same person credited for the discovery of penicillin<ref>Fleming, A. (1922) On a remarkable bacteriolytic element found in tissues and secretions. Proc.Roy.Soc.(London),93,306-317.</ref>. Discovery of the enzymatic activity was by accident; during the unrelated experiment, nasal drippings were inadvertently introduced to a petri dish containing a bacterial culture, which culture consequently exhibited the results of an as yet unknown enzymatic reaction. The observation of this unknown reaction led to further research on the components of this reaction as well as to the corresponding identification of the newfound "lysozyme." In 1965, David C. Phillips and coworkers determined the three-dimensional structure of lysozyme at 2 Å resolution <ref>PMID 5891407</ref>. Phillips' work was especially groundbreaking since Phillips had managed to successfully elucidate the structure of an enzyme via X-ray crystallography - a feat that had never before been accomplished<ref>Bugg, T. 1997. An Introduction to Enzyme and Coenzyme Chemistry. Blackwell Science Ltd., Oxford </ref><ref name="earliest">[http://www.umass.edu/microbio/rasmol/1st_xtls.htm Earliest Solutions for Macromolecular Crystal Structures].</ref>. Phillips' research also led to a structure-based hypothesis of its mechanism of action
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<ref name=Phillips1967>PMID 4382801</ref>.<br />
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'''Lysozyme''' - also known as muramidase, or glycoside hydrolase - is a powerful enzyme of biological significance found in abundance in tears, saliva, and human milk. In humans, it is encoded in the ''LYZ'' gene. Although it is responsible for the initial digestion of starches in the mouth, it is most widely identified as a non-specific defense in gram positive bacteria and in many species of fungi. Due to its antibacterial effects, it is a strong component of the innate immune system, and is an important part of an infant's diet to ward off diarrheal diseases. Since it is a small, easily available, and highly stable protein containing only 129 amino acid residues, it has been subject to extensive research regarding its function and structure. Hen Egg White (HEW) Lysozyme is shown below.
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==Function==
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The primary function of lysozyme is to hydrolyze the bonds between the sugar molecules in the peptidoglycan layer of bacterial cell walls. Peptidoglycan is a rigid structure that provides support and protection to bacterial cells. By cleaving these bonds, lysozyme weakens the bacterial cell wall, leading to the rupture and death of the bacterium.
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Lysozyme is particularly effective against Gram-positive bacteria, which have a relatively thick peptidoglycan layer. Gram-negative bacteria, on the other hand, have an outer membrane that provides additional protection, making them less susceptible to lysozyme.
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Lysozyme is an enzyme known for its unique ability to degrade the polysaccharide architecture of many kinds of cell walls, normally for the purpose of protection against bacterial infection<ref>Lysozyme. 2010. Citizendium.org. http://en.citizendium.org/wiki/Lysozyme</ref>. Its effects were first noticed by Laschtschenko in 1909. It was officially characterized and termed “lysozyme” by Alexander Fleming, the same person credited for the accidental discovery of penicillin.
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In addition to its antimicrobial activity, lysozyme also plays a role in other physiological processes. It contributes to the maintenance of healthy epithelial tissues, such as those lining the respiratory and gastrointestinal tracts. Lysozyme helps prevent bacterial overgrowth and infection in these areas by inhibiting the growth of certain bacteria.
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The characterization of lysozyme in 1922 by Alexander Fleming was providential in that the undertaken experiment related to the discovery of lysozyme was not geared toward any knowledge of such a protein as lysozyme <ref>Lysozyme. 2008. Lysozyme.co.uk. http://lysozyme.co.uk/</ref>. During the unrelated experiment, nasal drippings were inadvertently introduced to a petri dish containing a bacterial culture, which culture consequently exhibited the results of an as yet unknown enzymatic reaction. The observation of this unknown reaction led to further research on the components of this reaction as well as to the corresponding identification of the newfound "lysozyme." Fleming's discovery was complemented by David C. Phillips' 1965 description of the three-dimensional structure of lysozyme via a 200 pm resolution model obtained from X-ray crystallography <ref>Lysozyme, 2008. Lysozyme.co.uk. http://lysozyme.co.uk/</ref>. Phillips' work was especially groundbreaking since Phillips had managed to successfully elucidate the structure of an enzyme via X-ray crystallography - a feat that had never before been accomplished<ref>Bugg, T. 1997. An Introduction to Enzyme and Coenzyme Chemistry. Blackwell Science Ltd., Oxford </ref>. Phillips' research also led to the first sufficiently described enzymatic mechanism of catalytic action <ref>1967. Proc R Soc Lond B Bio 167 (1009): 389–401.</ref>. Thus, Phillips' elucidation of the function of lysozyme led Phillips to reach a more general conclusion on the diversity of enzymatic chemical action in relation to enzymatic structure. Clearly, the findings of Phillips as well as the more general historical development of the understanding of the structure and function of lysozyme have been paramount to the more general realm of enzyme chemistry. See also [[Molecular Playground/Lysozyme]] and [[User:Judy Voet/Lysozyme]].
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Lysozyme is widely distributed in nature and can be found in various organisms, including humans, animals, and plants. It is particularly abundant in the secretions of the lacrimal glands (tears) and salivary glands, where it helps protect the eyes and oral cavity from bacterial infections.
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==Function==
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Due to its antimicrobial properties, lysozyme has been used in various applications, such as a natural food preservative and an additive in certain personal care products. It has also been studied for its potential therapeutic applications, including in the development of antimicrobial agents and the treatment of certain infections.
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*'''Tail-associated lysozyme''' is expressed in bacteriophage T4 plays a role in digesting the peptidoglycan layering in lysis from within<ref>PMID: 3157805</ref>.
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==Chemical activity==
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[[Image:nag-nam2.jpg|left|400px|Lysozyme Cleavage Site<ref>Image from: http://www.vuw.ac.nz/staff/paul_teesdale-spittle/essentials/chapter-6/proteins/lysozyme.htm</ref>]]
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The particular substrate of preference for this cleavage type is a (NAG-NAM)₃ hexasaccharide, within which substrate occurs the
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cleaving target glycosidic bond, NAM₄-β-O-NAG₅. The individual hexasaccharide binding units are designated A-F, with NAM₄-β-O-NAG₅ glycosidic bond cleavage preference corresponding to a D-E unit glycosidic bond cl
Lysozyme is known for damaging bacterial cell walls by catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid (NAM) and N-acetyl-D-glucosamine (NAG) residues in peptidoglycan, and between N-acetyl-D-glucosamine residues in chitodextrins. In this way, lysozyme is efficient in lysing the cell walls of both bacteria and fungi. The location of cleavage for lysozyme on this architectural theme is the β(1-4) glycosidic linkage connecting the C1 carbon of NAM to the C4 carbon of NAG.
Lysozyme is known for damaging bacterial cell walls by catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid (NAM) and N-acetyl-D-glucosamine (NAG) residues in peptidoglycan, and between N-acetyl-D-glucosamine residues in chitodextrins. In this way, lysozyme is efficient in lysing the cell walls of both bacteria and fungi. The location of cleavage for lysozyme on this architectural theme is the β(1-4) glycosidic linkage connecting the C1 carbon of NAM to the C4 carbon of NAG.
The particular substrate of preference for this cleavage type is a (NAG-NAM)₃ hexasaccharide, within which substrate occurs the
The particular substrate of preference for this cleavage type is a (NAG-NAM)₃ hexasaccharide, within which substrate occurs the
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cleaving target glycosidic bond, NAM₄-β-O-NAG₅. The individual hexasaccharide binding units are designated A-F, with NAM₄-β-O-NAG₅ glycosidic bond cleavage preference corresponding to a D-E unit glycosidic bond cleavage preference.
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cleaving target glycosidic bond, NAM₄-β-O-NAG₅. The individual hexasaccharide binding units are designated A-F, with NAM₄-β-O-NAG₅ glycosidic bond cleavage preference corresponding to a D-E unit glycosidic bond cleavage preference. Depending on the organism from which lysozyme is obtained, hydrolysis of the glycosidic bond proceeds with retention of configuration at the anomeric carbon (hen egg white) or with inversion (goose, phage T4).
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{{clear}}
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[[Image:nag-nam2.jpg|thumb|left|400px|Lysozyme Cleavage Site<ref>Image from: http://www.vuw.ac.nz/staff/paul_teesdale-spittle/essentials/chapter-6/proteins/lysozyme.htm</ref>]]
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== Composition and Structure of Lysozyme ==
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All proteins consist of carbon, hydrogen, nitrogen, oxygen, and sulfur, as do most organic molecules. Enzymes are composed in such a way as to maximize their reactivity with their desired substrate, increasing the efficiency of biological reactions. The <scene name='Sandbox_39/Elements/1'>composition of lysozyme</scene> can be seen on the left, with the carbon atoms outlined in gray, oxygen atoms in red, nitrogen atoms in blue, sulfur atoms in yellow, and the three-letter abbreviation for the <scene name='Sandbox_39/Amino_acid_residues/1'>amino acid residues</scene> in purple. Lysozyme, like all proteins, also contains <scene name='Sandbox_39/C_and_n_terminal_residues/1'> C and N termini </scene>, and these can be seen by following the colors of the rainbow across the molecule. Starting at the red end, the C terminal end, one can work the entire way through to the N terminal end, showing the folding pattern and chain of the protein.
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Lysozyme efficiently acts on long (NAG-NAM) or (NAG) polymers. As the chain length gets smaller than six monomers, the catalytic rates drop substantially; in fact, trisaccharides act as competitive inhibitors by binding to the active site in a non-productive register.
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Lysozyme contains five <scene name='Sandbox_38/A/2'>alpha helical</scene> regions and five regions containing <scene name='Sandbox_38/B/1'>beta sheets</scene> as displayed in this <scene name='Sandbox_38/Alphab/1'>image</scene>. Linking these secondary structures, a number of beta turns and a large number of random coils make up the remainder of the polypeptide backbone. The polypeptide backbone of lysozyme involved in the 3 antiparallel beta sheets display the beta hairpin motif of supersecondary structure. This depiction of lysozyme contains an antiparallel beta-pleated sheet, which contributes greatly to the stability of the molecule by providing the correct alignment of hydrogen bonds. Lysozyme also contains a great deal of random coil, which is seen in the white regions of the molecule.
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<StructureSection load='' size='400' side='right' scene='37/376372/Overall/3' caption='Hen egg white lysozyme (PDB code [[1hew]])'>
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=== Intermolecular Interactions ===
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== Structure of Lysozyme ==
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'''Polarity'''
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The nature of the amino acid sidechains in the lysozyme polypeptide sequence leads to regions of varying hydrophobicities and polarities of the enzyme structure. The presence of certain regions of hydrophilicity and hydrophobicity is a driving force in determining protein structure when folding. The varying polarities of the side chains influence the locations of residues in the enzyme structure. Nonpolar residues appear blue, and polar residues appear red in the following <scene name='Sandbox_38/Non_polar_blue/1'>polarity</scene> display of lysozyme. Nonpolar residues will display hydrophobic tendencies occurring mostly on the interior of the enzyme while polar residues will increase in abundance on the surface of the protein in order to increase contact with the aqueous solvent so as to satisfy their hydrophilic nature. By observing a space-filled structural depiction of <scene name='Sandbox_38/Non_polar_blu/1'>lysozyme polarity</scene> with polar molecules colored red and nonpolar molecules colored blue the influence of polarity on protein folding is evident, with the blue (nonpolar) regions inside the red (polar) regions. The presence of <scene name='Hen_Egg-White_(HEW)_Lysozyme/Wuter/1'>water</scene> interacting with the various hydrophilic residues is depicted to further display how polarity affects structure. Water is depicted as yellow, and the polar and nonpolar regions remain their respective color.
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<jmol><jmolLink><script>script /scripts/37/376372/Overall/3.spt; hide water; set zshade off; spin off;</script><text>Lysozyme</text></jmolLink> </jmol> (shown here: PDB code 1HEW) is a small single-[[chain]] protein containing 129 [[amino acids]]. It folds into a compact structure with an active site cleft that binds to certain carbohydrates (<jmol><jmolLink><script> select ligand; selectionHalos ON; delay 0.5;selectionHalos OFF;</script><text>☼</text></jmolLink> </jmol>). Starting at the blue end, the N terminus(<jmol><jmolLink><script> select 1.CA; selectionHalos ON; delay 0.5;selectionHalos OFF;</script><text>☼</text></jmolLink> </jmol>), one can trace the entire chain (<jmol><jmolLink><script>select *.ca and 1; selectionhalos on;
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delay 0.5;
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select *.ca and 1-10; delay 0.3;
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select *.ca and 5-15; delay 0.2;
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select *.ca and 11-20; delay 0.1;
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select *.ca and 15-25; delay 0.1;
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select *.ca and 21-30; delay 0.1;
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select *.ca and 25-35; delay 0.1;
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select *.ca and 31-40; delay 0.1;
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select *.ca and 35-45; delay 0.1;
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select *.ca and 41-50; delay 0.1;
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select *.ca and 45-55; delay 0.1;
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select *.ca and 51-60; delay 0.1;
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select *.ca and 55-65; delay 0.1;
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select *.ca and 61-70; delay 0.1;
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select *.ca and 65-75; delay 0.1;
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select *.ca and 71-80; delay 0.1;
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select *.ca and 75-85; delay 0.1;
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select *.ca and 81-90; delay 0.1;
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select *.ca and 85-95; delay 0.1;
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select *.ca and 91-100; delay 0.1;
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select *.ca and 101-110; delay 0.1;
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select *.ca and 111-122; delay 0.1;
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select *.ca and 122-127; delay 0.2;
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select *.ca and 124-129; delay 0.3;
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select *.ca and 129; delay 0.5;
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selectionhalos off; </script><text>☼</text></jmolLink> </jmol>) through to the C terminus (<jmol><jmolLink><script> select 129.CA; selectionHalos ON; delay 0.5;selectionHalos OFF;</script><text>☼</text></jmolLink> </jmol>), showing the folding pattern of the protein. There are four disulfide bridges (between Cys 6 and Cys 127, between Cys 30 and Cys 115, between Cys 64 and Cys 80 and between Cys 76 and Cys 94 <jmol><jmolLink><script> select BONDS ({0:3}); wireframe 0.6; delay 0.5; wireframe 0.1</script><text>☼</text></jmolLink> </jmol>) that crosslink the polypeptide, stabilizing the native conformation. Lysozyme's secondary structure comprises four alpha (and one 3-10) helices (<jmol><jmolLink><script> select helix and *.CA; selectionHalos ON; delay 0.5;selectionHalos OFF;</script><text>☼</text></jmolLink> </jmol>)and five beta strands (<jmol><jmolLink><script> select sheet and *.CA; selectionHalos ON; delay 0.5;selectionHalos OFF;</script><text>☼</text></jmolLink> </jmol>). Linking these, a number of beta turns and a large number of random coils make up the remainder of the polypeptide backbone. The [[secondary structure]] is easier to see in a <scene name='37/376372/Secondary_structure/7'>ribbon view</scene>.
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'''Hydrogen Bonding'''
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However, this type of [[backbone representation]] ([https://stories.duke.edu/sciences-mother-of-ribbon-diagrams-celebrates-50-years-at-duke Richardson diagram]) was not invented until about a quarter century after this first enzyme structure was solved<ref name="jr1">PMID:10932243</ref>, so the figures in the original report look more like the previous scene.
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In all proteins <scene name='Sandbox_39/Hydrogen_bonds/2'>hydrogen bonds</scene> are essential for stability. In this ribbon diagram, the hydrogen bonds can be seen between the secondary structures of lysozyme highlighted in orange. Since the double bonds of the alpha carbons in the main chain of lysozyme cause torsional strain, lysozyme is limited to very specific hydrogen bonding between the amino acid residues. This representation clearly shows how crucial hydrogen bonding is to help maintain the stability of the protein.
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=== Surface properties ===
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'''Disulfide Bonding in Lysozyme'''
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Lysozyme contains four <scene name='Sandbox_39/Disulfide_bonds/1'>disulfide bonds</scene> involving eight cysteine residues, which are highlighted in yellow on the left. Disulfide bonds are intramolecular forces that stabilize the tertiary structure of many proteins. Disulfide bonds are present in four locations in lysozyme: between Cys 6 and Cys 127, between Cys 30 and Cys 115, between Cys 64 and Cys 80 and between Cys 76 and Cys 94.
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The shape and the properties of the <jmol><jmolLink><script> script /scripts/37/376372/Surface/4.spt; background white; hide water or ligand</script><text>surface of lysozyme</text></jmolLink> </jmol> determines what it binds to. When the protein folds into its native structure, the hydrophobicity and the charge of the side chains are a driving force. <jmol>
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<jmolLink>
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<script>select backbone or water; spacefill off; hide ligand or (sidechain and not (met, cys, ile, leu, val, phe, tyr, trp))</script>
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<text>Hydrophobic residues</text>
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</jmolLink>
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</jmol> (shown here: Met, Cys, Leu, Val, Ile, Phe, Tyr, and Trp) occur mostly on the interior of the enzyme while <jmol>
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<jmolLink>
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<script>select backbone or water; spacefill off; hide ligand or (sidechain and (met, cys, ile, leu, val, phe, tyr, trp))</script>
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<text>hydrophilic residues and proline</text>
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</jmolLink>
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</jmol> tend to be on the surface of the protein. In the crystal structure as well as in aqueous solution, <jmol>
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<jmolLink>
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<script>select backbone; spacefill on; select water; spacefill 1.0; display all</script>
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<text>water</text>
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</jmolLink></jmol> binds to the surface of the enzyme, as does the substrate and the product of the catalyzed hydrolysis reaction. To explore these aspects of protein structure and function, switch different parts of the structure on and off and change the coloring scheme (side chain polarity or charge) using the buttons below.
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== Mechanism ==
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<jmol>
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<item>
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<script>hide (hidden and not sidechain) or (sidechain and not (met, cys, ile, leu, val, phe, tyr, trp))</script>
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<text>hydrophobic side chains</text>
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</item>
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<item>
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<script>hide (hidden and not sidechain) or (sidechain and (met, cys, ile, leu, val, phe, tyr, trp))</script>
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<text>hydrophilic side chains</text>
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<script>display (displayed and not sidechain) or sidechain</script>
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<text>all side chains</text>
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<checked>true</checked>
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</jmolRadioGroup>
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</jmol>
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A ligand is able to bind to the active site of an enzyme to form a biologically relevant complex. The ligand can be seen in this
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<jmol>
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<scene name='Hen_Egg-White_(HEW)_Lysozyme/Ligand/1'>ribbon diagram</scene>, with the ligand protruding as a ball and stick model from the active site. Here, it is clear that the ligand is a polysaccharide.
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<jmolRadioGroup>
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<item>
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<script>select sidechain; color magenta; select sidechain and (cys, met, ile, leu, val, phe, tyr, trp); color gray; set echo ID bla 80% 0%; echo "hydrophobic"; color echo gray; frank off; set echo ID bla2 0% 0%; echo "hydrophilic"; color echo magenta </script>
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<text>colored by hydrophobiticity</text>
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<checked>true</checked>
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</item>
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<script>select sidechain; color white; select sidechain and (asp, glu); color red; select sidechain and (lys, arg, his); color blue; set echo ID bla 80% 0%; echo "positive"; color echo blue; frank off; set echo ID bla2 0% 0%; echo "negative"; color echo red</script>
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<text>colored by charge</text>
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<script> select sidechain; define ~consurf_to_do selected; define ~consurf_to_color selected; useFullScript = true; consurf_initial_scene = false; script /wiki/ConSurf/he/1hew_consurf.spt; set echo ID bla 80% 0%; echo "conserved"; color echo palevioletred; frank off; set echo ID bla2 0% 0%; echo "variable"; color echo darkturquoise</script>
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<text>colored by conservation</text>
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<checked>false</checked>
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</jmolRadioGroup>
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</jmol>
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The lysozyme reaction is characterized by hydrolysis of the beta (1-4) glycosidic bond between NAM and NAG. Lysozyme has a very specific active site, which can bind only six sugar rings from a polysaccharide chain. Once lysozyme binds to this chain, it hydrolyzes them. These six sugar rings represent the ligand of lysozyme. The lysozyme then distorts the fourth sugar in the six-membered complex, producing stress on the molecule and breaking the glycosidic bond.
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<jmol><jmolLink><script>if ({*.O and backbone and visible}.size>0) {select backbone; spacefill off} else {select backbone; spacefill on} </script><text>[± backbone spacefill]</text></jmolLink> </jmol>
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Lysozyme is best inhibited by small saccharides which act competitively with the natural substrate. The smaller saccharides will bind to the first three binding sites of the cleft (sites A-C), but will not reach sites D and E, where the enzyme cuts the glycosidic bond. So, the competitive inhibitor will stick in the cleft, not allowing the substrate to bind to the enzyme complex.<ref>http://mcdb-webarchive.mcdb.ucsb.edu/sears/biochemistry/tw-enz/lysozyme/HEWL/lysozyme-overview.htm</ref> Several known inhibitors of lysozyme are: SDS, N-acetyl-D-glucosamine, and various alcohols and oxidizing agents.<ref>http://www.worthington-biochem.com/ly/default.html</ref>
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<jmol><jmolLink><script>if ({ligand and hidden}.size>0) {display displayed or ligand} else {hide ligand or hidden} </script><text>[± ligand]</text></jmolLink> </jmol>
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[[Image:jrip.jpg|thumb|left|250px|Mechanism of Lysozyme<ref>Image from: http://www.google.com/imgres?imgurl=http://www.vuw.ac.nz/staff/paul_teesdale-spittle/essentials/chapter-6/pics-and-strucs/lysozyme-mech.gif&imgrefurl=http://www.vuw.ac.nz/staff/paul_teesdale-spittle/essentials/chapter-6/proteins/lysozyme.htm&usg=__ormapG4XKg-tR5GrMSOdSMTV4vE=&h=603&w=801&sz=7&hl=en&start=17&zoom=1&tbnid=nvr9gvFrUILDkM:&tbnh=143&tbnw=189&prev=/images%3Fq%3DThe%2Blysozyme%2Breaction%2Bmechanism%26um%3D1%26hl%3Den%26sa%3DN%26biw%3D1280%26bih%3D647%26tbs%3Disch:10%2C304&um=1&itbs=1&iact=hc&vpx=521&vpy=349&dur=448&hovh=191&hovw=254&tx=140&ty=48&ei=JQ_LTPKzLIjCsAPkzt2KDg&oei=IA_LTP74OsG78gapm-GFAQ&esq=2&page=2&ndsp=18&ved=1t:429,r:2,s:17&biw=1280&bih=647</ref>]]
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<jmol><jmolLink><script>if ({water and visible}.size>0) {select water; spacefill off} else {select water; spacefill 1.0} </script><text>[± water]</text></jmolLink> </jmol>
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The lysozyme mechanism of action results in the hydrolysis of a glycoside (hence the familial distinction of lysozyme as a glycosylase<ref>Lysozyme, 2008. Lysozyme.co.uk. http://lysozyme.co.uk/</ref>), which corresponds to the conversion of an acetal to a hemiacetal, which reaction (general degradation of glycosidic bond to units "capped" by newly formed hydroxyl groups) necessitates acid catalysis, since the conversion of acetal to hemiacetal involves the protonation of the reactant oxygen prior to actual bond cleavage. <ref>Pratt, C.W., Voet, D., Voet, J.G. Fundamentals of Biochemistry - Life at the Molecular Level - Third Edition. Voet, Voet and Pratt, 2008.</ref>. Furthermore, the transition state obtained from this protonation is a covalent, oxonium ion, intermediate that must obtain resonance stabilization. The need for some means of acid catalysis and covalent resonance stabilization is adequately provided by the Glu 35 and Asp 52 residues of lysozyme, respectively. The reaction mechanism of lysozyme is demonstrated on the left. The reaction begins at the upper left-hand side, and proceeds according to reaction arrows. Lysozyme works by hydrolyzing the glycosidic bond, distorting the bond between the NAM and NAG. This produces a glycosyl enzyme intermediate, which reacts with a water molecule to produce the product and the unchanged enzyme. The <scene name='Hen_Egg-White_(HEW)_Lysozyme/Act_site/2'>active site</scene> of lysozyme is formulated as a prominent cleft outlined by the two aforementioned catalytic amino acids, Glu 35 and Asp 52. The active site is geometrically bent to augment ligand binding, and the two amino acids interact with the ligand in the binding site. Asp52 is depicted in red, and Glu35 is depicted in green.
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===Hydrogen Bonding===
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== Applications of Lysozyme ==
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In all proteins <jmol><jmolLink>
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<script>set picking center; display all; script /scripts/37/376372/Hbonds/6.spt </script>
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<text>hydrogen bonds</text>
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</jmolLink></jmol> are essential for stability and function. Main chain hydrogen bonds are found within <jmol><jmolLink>
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<script>script /scripts/37/376372/Hbonds/1.spt; select helix and backbone; hbonds 0.2; delay 1.2; hbonds 0.04; </script>
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<text>helices</text>
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</jmolLink></jmol>, in <jmol><jmolLink>
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<script>script /scripts/37/376372/Hbonds/5.spt; select 74,77; hbonds 0.2; delay 1.2; hbonds 0.04; </script>
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<text>beta turns</text>
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</jmolLink></jmol> and <jmol><jmolLink>
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<script>script /scripts/37/376372/Hbonds/2.spt; select sheets and backbone; hbonds 0.2; delay 1.2; hbonds 0.04; </script>
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<text>between strands</text>
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</jmolLink></jmol>, connecting them to form sheets. <jmol>
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<jmolLink>
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<script>hide none; script /scripts/37/376372/Hbonds/4.spt </script>
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<text>Ordered waters</text>
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</jmolLink>
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</jmol> found in the crystal structure are bound to the protein surface by one or more hydrogen bonds. The specificity of the enzyme active site is partially determined by the hydrogen bonds <jmol>
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<text>between protein and ligand</text>
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</jmol>.
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Since lysozyme has been widely recognized for its antibacterial and antifungal properties, it has a wide variety of uses both in biochemical and pharmaceutical applications. In molecular biology, lysozyme is often used in the alkaline-lysis procedure for extracting and isolating plasmid DNA. It is used extensively in the pharmaceutical field for destroying gram-positive bacteria, and can be used to support already-existing immune defenses to fight bacterial infections. This enzyme is particularly important for preventing bacterial diseases in infants. Because of its antibacterial properties, lysozyme can also be used in the food industry to help prevent spoilage of foods.
+
This is the only scene that shows all atoms in the structure. Two tricks are used to improve the clarity of the scene. First, the atoms in the back (with respect to the current view) are faded. The same trick is used in this [https://cdn.rcsb.org/pdb101/geis/images/1000w/geis-0512-lysozyme.png painting] of the structure by Irving Geis. If you rotate the view to bring these atoms to the front, they will become bright and others will be faded. Second, the atoms in the very front are removed from view (slab). However, if you rotate the molecule they will come into view.
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== Structural history of Hen Egg-White Lysozyme==
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Lysozyme was the first enzyme whose X-ray structure was determined <ref> PMID 5840126</ref><ref>Phillips, D. C. The hen egg white lysozyme molecule. Proc. Natl Acad. Sci. USA 57, 483-495 (1967)</ref>. This <scene name='User:Judy_Voet/Lysozyme/Lysozyme1/15'>scene </scene> shows Hen Egg White (HEW) lysozyme containing a trisaccharide of N-acetylglucosamine (NAG) bound to a cleft in the enzyme. David Phillips, who determined the structure in 1965, saw that the cleft was large enough to fit three more saccharide units.
 
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He therefore built a model extending the trisaccharide to a
 
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<scene name='User:Judy_Voet/Lysozyme/Lysozyme1_hexamer/7'>hexasaccharide</scene> that fits into the cleft, labeling the sugar subsites A-F<ref> coordinates of the model kindly provided by Louise Johnson</ref>. Alternately click on <scene name='User:Judy_Voet/Lysozyme/Lysozyme1/15'>trisaccharide</scene> and <scene name='User:Judy_Voet/Lysozyme/Lysozyme1_hexamer/7'>hexasaccharide</scene> to turn the modeled portion of the hexasaccharide on and off.
 
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The interesting thing about the model was that the only way that the hexasaccharide would fit into the cleft was if the 4th saccharide (in subsite D) was strained into a <scene name='User:Judy_Voet/Lysozyme/Half-chair/2'>half-chair conformation</scene>. This conformation is what would be necessary for the formation of an oxocarbonium ion (oxionium ion). When the model was studied, <scene name='User:Judy_Voet/Lysozyme/Glu_35/1'>Glu 35</scene> was found to be in an ideal location to act as a general acid catalyst, 3.34 Angstroms from the bridging oxygen between the 4th and 5th saccharide units. <scene name='User:Judy_Voet/Lysozyme/Asp_52/2'>Asp 52</scene> appeared to be too far away (2.69 angstroms) in the static lysozyme structure to have formed a covalent bond with C1 of the half-chair model in the D site, and no covalent intermediate had ever been detected, so Phillips proposed that it acted as an electrostatic stabilizer of the oxonium ion (referred to as The Phillips Mechanism).
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== Ligand-bound structures ==
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Ligand-bound structures are valuable to understand specificity and mechanism of enzymes. Ideally, these structures include a substrate complex, a transition state analog complex and a product complex. Because lysozyme has a repetitive polymer as substrate and because of its specific binding properties, there is still no substrate complex structure with a glycosidic bond in the correct register. This has made it difficult to study the mechanism based on structural data (see below).
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<!--
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=== Inhibitor complex and hypothetical model of substrate complex ===
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<applet load='aln_1H6M_to_1HEW_2.pdb' size='350' frame='true' align='left' caption='NAG-2-deoxy-2-fluoro-glucosyl fluoride (NAG2FGlcF) bound to Glu35Gln HEW Lysozyme PDBid 1H6M' scene='User:Judy_Voet/Lysozyme/1h6m/3'/>
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-->
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Then, in 2001, Stephen Withers published <scene name='User:Judy_Voet/Lysozyme/1h6m/3'>1H6M</scene>,<ref>PMID 11518970</ref> in which Glu 35 had been mutated to Gln to remove the general acid catalyst. The substrate contained NAG-2-fluoro-glucosyl fluoride (NAG2FGlcF). The fluoro group on C-1 does not require acid catalysis to be a good leaving group, and the remaining saccharide, in the absence of the acid necessary to catalyse the second step of the reaction, was demonstrated to form a <scene name='User:Judy_Voet/Lysozyme/Covalent/1'> covalent intermediate</scene>. In this <scene name='User:Judy_Voet/Lysozyme/Superposition/2'>superposition</scene> of the half chair model with 1HEW (greens) and the covalent intermediate in 1H6M (blues), note the relatively small motions of Asp 52 and C1 of the sugar ring in going from the model to the covalent intermediate. to observe the motion from the <scene name='User:Judy_Voet/Lysozyme/Asp52_halfchair/1'>half-chair</scene> to the <scene name='User:Judy_Voet/Lysozyme/Covalent/2'>covalent intermediate</scene> just toggle between the two green links.
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Lysozyme is inhibited by short-length oligosaccharides which act competitively with the natural substrate. The smaller saccharides will bind to the first three binding sites of the cleft (sites A-C), but will not reach sites D and E, where the enzyme cuts the glycosidic bond. So the competitive inhibitor will stick in the cleft, not allowing the substrate to bind to the enzyme complex.<ref>http://mcdb-webarchive.mcdb.ucsb.edu/sears/biochemistry/tw-enz/lysozyme/HEWL/lysozyme-overview.htm</ref>. When the 2 Å structure of lysozyme was reported in 1965<ref> PMID 5891407</ref>, it was published back to back with a report of several 6 Å structures of inhibitor-bound structures<ref> PMID 5840126</ref>. The refined structure of one of these inhibitor structures at higher resolution was deposited as 1HEW in the protein data bank in 1992, and this is the structure all the previous scenes are based on. The bound inhibitor (NAG)<sub>3</sub> occupies subsites A, B, and C.
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In order to deduce the catalytic mechanism of lysozyme, the Phillips groups modeled six sugar residues into the active site. Phillips describes <scene name='37/376372/Hex_model/1'>this hypothetical model</scene> in a 1966 article in Scientific American<ref>Phillips (1966) Scientific American 215, 76-90</ref>, and it is featured as a painting by Irving Geis on the [https://pdb101.rcsb.org/sci-art/geis-archive/gallery/geis-0512-lysozyme front cover].
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<jmol>
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=== Covalent intermediate and product complex ===
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In 2001, Stephen Withers and coworkers published a <scene name='37/376372/Covalent_intermediate/2'>covalent intermediate</scene> (PDB code 1H6M) in which Glu 35 had been mutated to Gln to remove the general acid catalyst and the substrate contained NAG-2-fluoro-glucosyl fluoride (NAG2FGlcF) <ref name=Withers2001>PMID 11518970</ref>. The combination of these two changes slowed down the reaction sufficiently to capture the intermediate. This structure and the biochemical results were strong evidence that lysozyme forms a covalent intermediate (via Asp 52) as one step in the reaction.
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When lysozyme breaks down peptidoglycan, the product of the reaction is a shortened peptidoglycan that is broken down further by lysozyme. This makes it difficult to crystallize an enzyme: product complex. Vocadlo and coworkers <ref>Davies, Withers and Vocadlo (2009) The Chitopentaose Complex of a Mutant Hen Egg-White Lysozyme Displays No Distortion of the –1 Sugar Away from a 4C1 Chair Conformation, Australian Journal of Chemistry 62(6) 528-532</ref> determined the structure of a <scene name='37/376372/Product_complex/6'>product complex with five sugar units</scene> (PDB code 2WAR) by cocrystallizing the oligosaccharide with an almost inactive mutant of lysozyme (using the same Glu35Gln mutant as used for the covalent intermediate). To compare with the covalent intermediate structure, use the selections below:
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<text>view 1</text>
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<text>🔎 active site</text>
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<text>🔎 active site 2</text>
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==Mechanism==
 +
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Hydrolysis of glycosidic bonds by hen egg white lysozyme proceeds with retention of configuration. In 1953, Koshland <ref>Koshland, D. E. (1953). Biol. Rev. 28, 416–436</ref> suggested that in general, retention of configuration implies a double-displacement mechanism (while inversion of configuration implies single displacement). For decades, two competing mechanistic hypotheses (Phillips<ref name=Phillips1967/>: dissociative mechanism with oxocarbenium intermediate; Koshland: two-step associative mechanism with covalent enzyme complex as intermediate) were considered, with data from 2001<ref name=Withers2001/> tipping the scale toward the existence of a covalent intermediate. The absence of a substrate complex structure certainly contributed to difficulties distinguishing between possible mechanisms, as did the existence of two distinct mechanisms (retention and inversion of configuration) within the same structural family of enzymes (e.g. hen vs goose enzyme).
 +
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[[Image:Mechanism_of_Lysozyme_action.png|left|800px|Mechanism of Lysozyme]]
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{{Clear}}
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Lysozyme hydrolyzes a glycoside (hence the familial classification of lysozyme as a glycosylase<ref>https://bio.libretexts.org/Bookshelves/Biochemistry/Book%3A_Biochemistry_Online_(Jakubowski)/07%3A_CATALYSIS/B._Mechanisms_of_Enzyme-Catalyzed_Reactions/B2.__Lysozyme</ref>), which corresponds to the conversion of an acetal to a hemiacetal. The reaction proceeds in two steps as shown in the figure above. In the first step, Asp 52 acts as nucleophile and part of the sugar is the leaving group. In the second step, water acts as nucleophile and Asp 52 acts as leaving group. Both steps invert the configuration at the anomeric carbon, leading to an overall retention of configuration. Glu 35 acts as an acid in the first step (protonating the sugar the the glycosidic bond to make it a better electrophile) and as a base in the second step (deprotonating water to make it a better nucleophile). While the figure shows some of the sugars in a boat conformation to emphasize the inversion of configuration, these are not observed experimentally but are rather found in a chair conformation.
 +
 +
== Applications of Lysozyme ==
 +
 +
Since lysozyme has been widely recognized for its antibacterial and antifungal properties, it has a wide variety of uses both in biochemical and pharmaceutical applications. In molecular biology, lysozyme is often used in the alkaline-lysis procedure for extracting and isolating plasmid DNA. It is used extensively in the pharmaceutical field for destroying gram-positive bacteria, and can be used to support already-existing immune defenses to fight bacterial infections. This enzyme is particularly important for preventing bacterial diseases in infants. Because of its antibacterial properties, lysozyme can also be used in the food industry to help prevent spoilage of foods.
 +
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==3D structures of lysozome==
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[[Lysozyme 3D structures]]
== See Also ==
== See Also ==
-
* [[Lysozyme 3D structures]]
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* [http://en.wikipedia.org/wiki/Lysozyme Lysozyme]
* [http://en.wikipedia.org/wiki/Lysozyme Lysozyme]
* [http://en.wikipedia.org/wiki/Glycoside_hydrolase#Retaining_glycoside_hydrolases Retaining Glycoside Hydrolases]
* [http://en.wikipedia.org/wiki/Glycoside_hydrolase#Retaining_glycoside_hydrolases Retaining Glycoside Hydrolases]
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*[[Molecular Playground/Lysozyme]]
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*[[User:Judy Voet/Lysozyme]]
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*[[Lysozyme (Arabic)]]
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*[[Lysozyme (hebrew)]]
== References ==
== References ==
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[[Category:Topic Page]]
[[Category:Topic Page]]
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[[ar: Lysozyme (Arabic)]]
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[[he: Lysozyme (hebrew)]]

Current revision

Contents

Hen egg-white lysozyme, 1HEW (scroll down to the structure section for interactive figure)
Hen egg-white lysozyme, 1HEW (scroll down to the structure section for interactive figure)

Lysozyme - also known as muramidase - is a powerful enzyme found in abundance in tears, saliva, and human milk. In humans, it is encoded in the LYZ gene. Since it is a small, easily available, and highly stable protein, it has been subject to extensive research regarding its function and structure.

Introduction

Lysozyme acts as a non-specific defense against bacteria and fungi. It is a component of the innate immune system, and is an important part of an infant's diet to ward off diarrhea. It is an enzyme known for its ability to degrade the polysaccharide architecture of many kinds of cell walls, normally for the purpose of protection against bacterial infection[1]. The structure of hen egg white (HEW) lysozyme, the focus of this article, is shown on the right. The antibacterial activity of hen egg white was first described by Laschtschenko in 1909[2]. It was characterized and named “lysozyme” by Alexander Fleming, the same person credited for the discovery of penicillin[3]. Discovery of the enzymatic activity was by accident; during the unrelated experiment, nasal drippings were inadvertently introduced to a petri dish containing a bacterial culture, which culture consequently exhibited the results of an as yet unknown enzymatic reaction. The observation of this unknown reaction led to further research on the components of this reaction as well as to the corresponding identification of the newfound "lysozyme." In 1965, David C. Phillips and coworkers determined the three-dimensional structure of lysozyme at 2 Å resolution [4]. Phillips' work was especially groundbreaking since Phillips had managed to successfully elucidate the structure of an enzyme via X-ray crystallography - a feat that had never before been accomplished[5][6]. Phillips' research also led to a structure-based hypothesis of its mechanism of action [7].

Function

The primary function of lysozyme is to hydrolyze the bonds between the sugar molecules in the peptidoglycan layer of bacterial cell walls. Peptidoglycan is a rigid structure that provides support and protection to bacterial cells. By cleaving these bonds, lysozyme weakens the bacterial cell wall, leading to the rupture and death of the bacterium.

Lysozyme is particularly effective against Gram-positive bacteria, which have a relatively thick peptidoglycan layer. Gram-negative bacteria, on the other hand, have an outer membrane that provides additional protection, making them less susceptible to lysozyme.

In addition to its antimicrobial activity, lysozyme also plays a role in other physiological processes. It contributes to the maintenance of healthy epithelial tissues, such as those lining the respiratory and gastrointestinal tracts. Lysozyme helps prevent bacterial overgrowth and infection in these areas by inhibiting the growth of certain bacteria.

Lysozyme is widely distributed in nature and can be found in various organisms, including humans, animals, and plants. It is particularly abundant in the secretions of the lacrimal glands (tears) and salivary glands, where it helps protect the eyes and oral cavity from bacterial infections.

Due to its antimicrobial properties, lysozyme has been used in various applications, such as a natural food preservative and an additive in certain personal care products. It has also been studied for its potential therapeutic applications, including in the development of antimicrobial agents and the treatment of certain infections.

  • Tail-associated lysozyme is expressed in bacteriophage T4 plays a role in digesting the peptidoglycan layering in lysis from within[8].

Chemical activity

Lysozyme Cleavage Site

The particular substrate of preference for this cleavage type is a (NAG-NAM)₃ hexasaccharide, within which substrate occurs the cleaving target glycosidic bond, NAM₄-β-O-NAG₅. The individual hexasaccharide binding units are designated A-F, with NAM₄-β-O-NAG₅ glycosidic bond cleavage preference corresponding to a D-E unit glycosidic bond cl

Lysozyme is known for damaging bacterial cell walls by catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid (NAM) and N-acetyl-D-glucosamine (NAG) residues in peptidoglycan, and between N-acetyl-D-glucosamine residues in chitodextrins. In this way, lysozyme is efficient in lysing the cell walls of both bacteria and fungi. The location of cleavage for lysozyme on this architectural theme is the β(1-4) glycosidic linkage connecting the C1 carbon of NAM to the C4 carbon of NAG.

The particular substrate of preference for this cleavage type is a (NAG-NAM)₃ hexasaccharide, within which substrate occurs the cleaving target glycosidic bond, NAM₄-β-O-NAG₅. The individual hexasaccharide binding units are designated A-F, with NAM₄-β-O-NAG₅ glycosidic bond cleavage preference corresponding to a D-E unit glycosidic bond cleavage preference. Depending on the organism from which lysozyme is obtained, hydrolysis of the glycosidic bond proceeds with retention of configuration at the anomeric carbon (hen egg white) or with inversion (goose, phage T4).

Lysozyme efficiently acts on long (NAG-NAM) or (NAG) polymers. As the chain length gets smaller than six monomers, the catalytic rates drop substantially; in fact, trisaccharides act as competitive inhibitors by binding to the active site in a non-productive register.

Hen egg white lysozyme (PDB code 1hew)

Drag the structure with the mouse to rotate


Mechanism

Hydrolysis of glycosidic bonds by hen egg white lysozyme proceeds with retention of configuration. In 1953, Koshland [17] suggested that in general, retention of configuration implies a double-displacement mechanism (while inversion of configuration implies single displacement). For decades, two competing mechanistic hypotheses (Phillips[7]: dissociative mechanism with oxocarbenium intermediate; Koshland: two-step associative mechanism with covalent enzyme complex as intermediate) were considered, with data from 2001[15] tipping the scale toward the existence of a covalent intermediate. The absence of a substrate complex structure certainly contributed to difficulties distinguishing between possible mechanisms, as did the existence of two distinct mechanisms (retention and inversion of configuration) within the same structural family of enzymes (e.g. hen vs goose enzyme).


Mechanism of Lysozyme

Lysozyme hydrolyzes a glycoside (hence the familial classification of lysozyme as a glycosylase[18]), which corresponds to the conversion of an acetal to a hemiacetal. The reaction proceeds in two steps as shown in the figure above. In the first step, Asp 52 acts as nucleophile and part of the sugar is the leaving group. In the second step, water acts as nucleophile and Asp 52 acts as leaving group. Both steps invert the configuration at the anomeric carbon, leading to an overall retention of configuration. Glu 35 acts as an acid in the first step (protonating the sugar the the glycosidic bond to make it a better electrophile) and as a base in the second step (deprotonating water to make it a better nucleophile). While the figure shows some of the sugars in a boat conformation to emphasize the inversion of configuration, these are not observed experimentally but are rather found in a chair conformation.

Applications of Lysozyme

Since lysozyme has been widely recognized for its antibacterial and antifungal properties, it has a wide variety of uses both in biochemical and pharmaceutical applications. In molecular biology, lysozyme is often used in the alkaline-lysis procedure for extracting and isolating plasmid DNA. It is used extensively in the pharmaceutical field for destroying gram-positive bacteria, and can be used to support already-existing immune defenses to fight bacterial infections. This enzyme is particularly important for preventing bacterial diseases in infants. Because of its antibacterial properties, lysozyme can also be used in the food industry to help prevent spoilage of foods.

3D structures of lysozome

Lysozyme 3D structures

See Also

References

  1. ↑ Ragland SA, Criss AK. From bacterial killing to immune modulation: Recent insights into the functions of lysozyme. PLoS Pathog. 2017 Sep 21;13(9):e1006512. doi: 10.1371/journal.ppat.1006512., eCollection 2017 Sep. PMID:28934357 doi:http://dx.doi.org/10.1371/journal.ppat.1006512
  2. ↑ Laschtschenko,P. (1909) Über die keimtötende und entwicklungshemmende Wirkung von Hühnereiweiss. Z. Hyg. Infektionskrankh.,64,419-427.
  3. ↑ Fleming, A. (1922) On a remarkable bacteriolytic element found in tissues and secretions. Proc.Roy.Soc.(London),93,306-317.
  4. ↑ Blake CC, Koenig DF, Mair GA, North AC, Phillips DC, Sarma VR. Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 Angstrom resolution. Nature. 1965 May 22;206(986):757-61. PMID:5891407
  5. ↑ Bugg, T. 1997. An Introduction to Enzyme and Coenzyme Chemistry. Blackwell Science Ltd., Oxford
  6. ↑ Earliest Solutions for Macromolecular Crystal Structures.
  7. ↑ 7.0 7.1 Blake CC, Johnson LN, Mair GA, North AC, Phillips DC, Sarma VR. Crystallographic studies of the activity of hen egg-white lysozyme. Proc R Soc Lond B Biol Sci. 1967 Apr 18;167(1009):378-88. doi:, 10.1098/rspb.1967.0035. PMID:4382801 doi:http://dx.doi.org/10.1098/rspb.1967.0035
  8. ↑ Nakagawa H, Arisaka F, Ishii S. Isolation and characterization of the bacteriophage T4 tail-associated lysozyme. J Virol. 1985 May;54(2):460-6. PMID:3157805 doi:10.1128/JVI.54.2.460-466.1985
  9. ↑ Image from: http://www.vuw.ac.nz/staff/paul_teesdale-spittle/essentials/chapter-6/proteins/lysozyme.htm
  10. ↑ Richardson JS. Early ribbon drawings of proteins. Nat Struct Biol. 2000 Aug;7(8):624-5. doi: 10.1038/77912. PMID:10932243 doi:http://dx.doi.org/10.1038/77912
  11. ↑ http://mcdb-webarchive.mcdb.ucsb.edu/sears/biochemistry/tw-enz/lysozyme/HEWL/lysozyme-overview.htm
  12. ↑ Blake CC, Koenig DF, Mair GA, North AC, Phillips DC, Sarma VR. Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 Angstrom resolution. Nature. 1965 May 22;206(986):757-61. PMID:5891407
  13. ↑ Johnson LN, Phillips DC. Structure of some crystalline lysozyme-inhibitor complexes determined by X-ray analysis at 6 Angstrom resolution. Nature. 1965 May 22;206(986):761-3. PMID:5840126
  14. ↑ Phillips (1966) Scientific American 215, 76-90
  15. ↑ 15.0 15.1 Vocadlo DJ, Davies GJ, Laine R, Withers SG. Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature. 2001 Aug 23;412(6849):835-8. PMID:11518970 doi:10.1038/35090602
  16. ↑ Davies, Withers and Vocadlo (2009) The Chitopentaose Complex of a Mutant Hen Egg-White Lysozyme Displays No Distortion of the –1 Sugar Away from a 4C1 Chair Conformation, Australian Journal of Chemistry 62(6) 528-532
  17. ↑ Koshland, D. E. (1953). Biol. Rev. 28, 416–436
  18. ↑ https://bio.libretexts.org/Bookshelves/Biochemistry/Book%3A_Biochemistry_Online_(Jakubowski)/07%3A_CATALYSIS/B._Mechanisms_of_Enzyme-Catalyzed_Reactions/B2.__Lysozyme
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