Lysozyme (EC 3.2.1.17, muramidase, ''N''-acetylmuramide glycanhydrolase; systematic name peptidoglycan ''N''-acetylmuramoylhydrolase) is an
antimicrobial
An antimicrobial is an agent that kills microorganisms or stops their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, antibiotics are used against bacteria, and antifungals ar ...
enzyme produced by animals that forms part of the
innate immune system. It is a
glycoside hydrolase
Glycoside hydrolases (also called glycosidases or glycosyl hydrolases) catalyze the hydrolysis of glycosidic bonds in complex sugars. They are extremely common enzymes with roles in nature including degradation of biomass such as cellulose (ce ...
that catalyzes the following process:
: Hydrolysis of (1→4)-β-linkages between ''N''-acetylmuramic acid and ''N''-acetyl-
D-glucosamine residues in a peptidoglycan and between ''N''-acetyl-
D-glucosamine residues in chitodextrins
Peptidoglycan is the major component of
gram-positive bacterial cell wall.
This hydrolysis in turn compromises the integrity of bacterial cell walls causing
lysis of the bacteria.
Lysozyme is abundant in
secretions including
tears
Tears are a clear liquid secreted by the lacrimal glands (tear gland) found in the eyes of all land mammals. Tears are made up of water, electrolytes, proteins, lipids, and mucins that form layers on the surface of eyes. The different types of ...
,
saliva,
human milk
Breast milk (sometimes spelled as breastmilk) or mother's milk is milk produced by mammary glands located in the breast of a human female. Breast milk is the primary source of nutrition for newborns, containing fat, protein, carbohydrates (lacto ...
, and
mucus
Mucus ( ) is a slippery aqueous secretion produced by, and covering, mucous membranes. It is typically produced from cells found in mucous glands, although it may also originate from mixed glands, which contain both serous and mucous cells. It ...
. It is also present in
cytoplasmic granules of the
macrophages
Macrophages (abbreviated as M φ, MΦ or MP) ( el, large eaters, from Greek ''μακρός'' (') = large, ''φαγεῖν'' (') = to eat) are a type of white blood cell of the immune system that engulfs and digests pathogens, such as cancer ce ...
and the
polymorphonuclear neutrophil
Neutrophils (also known as neutrocytes or heterophils) are the most abundant type of granulocytes and make up 40% to 70% of all white blood cells in humans. They form an essential part of the innate immune system, with their functions varying in ...
s (PMNs). Large amounts of lysozyme can be found in
egg white. C-type lysozymes are closely related to
α-lactalbumin in sequence and structure, making them part of the same
glycoside hydrolase family 22
In molecular biology, glycoside hydrolase family 22 is a Protein family, family of glycoside hydrolases.
Glycoside hydrolases are a widespread group of enzymes that hydrolyse the glycosidic bond between two or more carbohydrates, or between a ca ...
. In humans, the C-type lysozyme enzyme is encoded by the ''LYZ'' gene.
Hen egg white lysozyme is thermally stable, with a
melting point
The melting point (or, rarely, liquefaction point) of a substance is the temperature at which it changes state from solid to liquid. At the melting point the solid and liquid phase exist in equilibrium. The melting point of a substance depen ...
reaching up to 72 °C at pH 5.0. However, lysozyme in human milk loses activity very quickly at that temperature. Hen egg white lysozyme maintains its activity in a large range of pH (6–9). Its
isoelectric point
The isoelectric point (pI, pH(I), IEP), is the pH at which a molecule carries no net electrical charge or is electrically neutral in the statistical mean. The standard nomenclature to represent the isoelectric point is pH(I). However, pI is also u ...
is 11.35. The isoelectric point of human milk lysozyme is 10.5–11.
Function and mechanism
The
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
functions by hydrolyzing glycosidic bonds in
peptidoglycan
Peptidoglycan or murein is a unique large macromolecule, a polysaccharide, consisting of sugars and amino acids that forms a mesh-like peptidoglycan layer outside the plasma membrane, the rigid cell wall (murein sacculus) characteristic of most ba ...
s. The enzyme can also break
glycosidic bond
A glycosidic bond or glycosidic linkage is a type of covalent bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate.
A glycosidic bond is formed between the hemiacetal or hemiketal group ...
s in
chitin, although not as effectively as true
chitinase
Chitinases (EC 3.2.1.14, chitodextrinase, 1,4-β-poly-N-acetylglucosaminidase, poly-β-glucosaminidase, β-1,4-poly-N-acetyl glucosamidinase, poly ,4-(N-acetyl-β-D-glucosaminide)glycanohydrolase, (1→4)-2-acetamido-2-deoxy-β-D-glucan glycano ...
s.
![Mecanism of action for Lysozyme](https://upload.wikimedia.org/wikipedia/commons/2/2f/Mecanism_of_action_for_Lysozyme.svg)
Lysozyme's active site binds the
peptidoglycan
Peptidoglycan or murein is a unique large macromolecule, a polysaccharide, consisting of sugars and amino acids that forms a mesh-like peptidoglycan layer outside the plasma membrane, the rigid cell wall (murein sacculus) characteristic of most ba ...
molecule in the prominent cleft between its two domains. It attacks peptidoglycans (found in the cell walls of bacteria, especially
Gram-positive bacteria), its natural substrate, between
''N''-acetylmuramic acid (NAM) and the fourth carbon atom of
N-acetylglucosamine (NAG).
Shorter
saccharides
In organic chemistry, a carbohydrate () is a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms, usually with a hydrogen–oxygen atom ratio of 2:1 (as in water) and thus with the empirical formula (where ''m'' may or may ...
like tetrasaccharide have also shown to be viable substrates but via an intermediate with a longer chain. Chitin has also been shown to be a viable lysozyme substrate. Artificial substrates have also been developed and used in lysozyme.
Mechanism
Phillips
The Phillips mechanism proposed that the enzyme's catalytic power came from both steric strain on the bound substrate and electrostatic stabilization of an
oxo-carbenium intermediate. From X-ray crystallographic data, Phillips proposed the active site of the enzyme, where a hexasaccharide binds. The lysozyme distorts the fourth sugar (in the D or -1 subsite) in the hexasaccharide into a half-chair conformation. In this stressed state, the glycosidic bond is more easily broken. An ionic intermediate containing an
oxo-carbenium is created as a result of the glycosidic bond breaking.
Thus distortion causing the substrate molecule to adopt a strained conformation similar to that of the transition state will lower the energy barrier of the reaction.
The proposed oxo-carbonium intermediate was speculated to be electrostatically stabilized by aspartate and glutamate residues in the active site by
Arieh Warshel
Arieh Warshel ( he, אריה ורשל; born November 20, 1940) is an Israeli-American biochemist and biophysicist. He is a pioneer in computational studies on functional properties of biological molecules, Distinguished Professor of Chemistry ...
in 1978. The electrostatic stabilization argument was based on comparison to bulk water, the reorientation of water dipoles can cancel out the stabilizing energy of charge interaction. In Warshel's model, the enzyme acts as a super-solvent, which fixes the orientation of ion pairs and provides super-
solvation
Solvation (or dissolution) describes the interaction of a solvent with dissolved molecules. Both ionized and uncharged molecules interact strongly with a solvent, and the strength and nature of this interaction influence many properties of the ...
(very good stabilization of ion pairs), and especially lower the energy when two ions are close to each other.
The
rate-determining step
In chemical kinetics, the overall rate of a reaction is often approximately determined by the slowest step, known as the rate-determining step (RDS or RD-step or r/d step) or rate-limiting step. For a given reaction mechanism, the prediction of the ...
(RDS) in this mechanism is related to formation of the
oxo-carbenium intermediate. There were some contradictory results to indicate the exact RDS. By tracing the formation of product (
p-nitrophenol), it was discovered that the RDS can change over different temperatures, which was a reason for those contradictory results. At a higher temperature the RDS is formation of glycosyl enzyme intermediate and at a lower temperature the break down of that intermediate.
Covalent mechanism
![LysozymeIntermediates copy](https://upload.wikimedia.org/wikipedia/commons/4/41/LysozymeIntermediates_copy.png)
In an early debate in 1969, Dahlquist proposed a covalent mechanism for lysozyme based on
kinetic isotope effect
In physical organic chemistry, a kinetic isotope effect (KIE) is the change in the reaction rate of a chemical reaction when one of the atoms in the reactants is replaced by one of its isotopes. Formally, it is the ratio of rate constants for th ...
,
but for a long time the ionic mechanism was more accepted. In 2001, a revised mechanism was proposed by Vocadlo via a covalent but not ionic intermediate. Evidence from
ESI-
MS analysis indicated a covalent intermediate. A 2-fluoro substituted substrate was used to lower the reaction rate and accumulate an intermediate for characterization.
The amino acid side-chains glutamic acid 35 (Glu35) and aspartate 52 (Asp52) have been found to be critical to the activity of this enzyme. Glu35 acts as a proton donor to the glycosidic bond, cleaving the C-O bond in the substrate, whereas Asp52 acts as a
nucleophile to generate a glycosyl enzyme intermediate. The Glu35 reacts with water to form hydroxyl ion, a stronger
nucleophile than water, which then attacks the glycosyl enzyme intermediate, to give the product of hydrolysis and leaving the enzyme unchanged.
This type of covalent mechanism for enzyme catalysis was first proposed by
Koshland.
More recently, quantum mechanics/ molecular mechanics (QM/MM)
molecular dynamics
Molecular dynamics (MD) is a computer simulation method for analyzing the physical movements of atoms and molecules. The atoms and molecules are allowed to interact for a fixed period of time, giving a view of the dynamic "evolution" of t ...
simulations have been using the crystal of HEWL and predict the existence of a covalent intermediate.
Evidence for the ESI-MS and X-ray structures indicate the existence of covalent intermediate, but primarily rely on using a less active mutant or non-native substrate. Thus, QM/MM molecular dynamics provides the unique ability to directly investigate the mechanism of wild-type HEWL and native substrate. The calculations revealed that the covalent intermediate from the covalent mechanism is ~30 kcal/mol more stable than the ionic intermediate from the Phillips mechanism.
These calculation demonstrate that the ionic intermediate is extremely energetically unfavorable and the covalent intermediates observed from experiments using less active mutant or non-native substrates provide useful insight into the mechanism of wild-type HEWL.
Inhibition
Imidazole
Imidazole (ImH) is an organic compound with the formula C3N2H4. It is a white or colourless solid that is soluble in water, producing a mildly alkaline solution. In chemistry, it is an aromatic heterocycle, classified as a diazole, and has non-a ...
derivatives can form a
charge-transfer complex with some residues (in or outside active center) to achieve a competitive inhibition of lysozyme. In
Gram-negative bacteria
Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall ...
, the
lipopolysaccharide
Lipopolysaccharides (LPS) are large molecules consisting of a lipid and a polysaccharide that are bacterial toxins. They are composed of an O-antigen, an outer core, and an inner core all joined by a covalent bond, and are found in the outer ...
acts as a non-competitive inhibitior by highly favored binding with lysozyme.
Non-enzymatic action
Despite that the muramidase activity of lysozyme has been supposed to play the key role for its antibacterial properties, evidence of its non-enzymatic action was also reported. For example, blocking the catalytic activity of lysozyme by mutation of critical amino acid in the active site (52-
Asp -> 52-
Ser) does not eliminate its antimicrobial activity. The lectin-like ability of lysozyme to recognize bacterial carbohydrate antigen without lytic activity was reported for tetrasaccharide related to
lipopolysaccharide
Lipopolysaccharides (LPS) are large molecules consisting of a lipid and a polysaccharide that are bacterial toxins. They are composed of an O-antigen, an outer core, and an inner core all joined by a covalent bond, and are found in the outer ...
of ''
Klebsiella pneumoniae
''Klebsiella pneumoniae'' is a Gram-negative, non-motile, encapsulated, lactose- fermenting, facultative anaerobic, rod-shaped bacterium. It appears as a mucoid lactose fermenter on MacConkey agar.
Although found in the normal flora of the mo ...
''. Also, lysozyme interacts with antibodies and
T-cell receptors.
Enzyme conformation changes
Lysozyme exhibits two conformations: an open active state and a closed inactive state. The catalytic relevance was examined with single walled
carbon nanotubes (SWCN) field effect transistors (FETs), where a singular lysozyme was bound to the SWCN FET. Electronically monitoring the lysozyme showed two conformations, an open active site and a closed inactive site. In its active state lysozyme is able to
processively hydrolyze its substrate, breaking on average 100 bonds at a rate of 15 per second. In order to bind a new substrate and move from the closed inactive state to the open active state requires two conformation step changes, while inactivation requires one step.
Role in disease and therapy
Lysozyme is part of the innate immune system. Reduced lysozyme levels have been associated with
bronchopulmonary dysplasia
Bronchopulmonary dysplasia (BPD; part of the spectrum of chronic lung disease of infancy) is a chronic lung disease in which premature infants, usually those who were treated with supplemental oxygen, require long-term oxygen. The alveoli that are ...
in newborns.
Piglets fed with human lysozyme milk can recover from diarrheal disease caused by ''E. coli'' faster. The concentration of lysozyme in human milk is 1,600 to 3,000 times greater than the concentration in livestock milk. Human lysozyme is more active than hen egg white lysozyme. A
transgenic
A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the ...
line of goats (with a
founder
Founder or Founders may refer to:
Places
*Founders Park, a stadium in South Carolina, formerly known as Carolina Stadium
* Founders Park, a waterside park in Islamorada, Florida
Arts, entertainment, and media
* Founders (''Star Trek''), the ali ...
named "Artemis") were developed to produce milk with human lysozyme to protect children from diarrhea if they can't get the benefits of human breastfeeding.
Since lysozyme is a natural form of protection from
Gram-positive
In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall.
Gram-positive bact ...
pathogens like ''
Bacillus
''Bacillus'' (Latin "stick") is a genus of Gram-positive, rod-shaped bacteria, a member of the phylum '' Bacillota'', with 266 named species. The term is also used to describe the shape (rod) of other so-shaped bacteria; and the plural ''Bacill ...
'' and ''
Streptococcus
''Streptococcus'' is a genus of gram-positive ' (plural ) or spherical bacteria that belongs to the family Streptococcaceae, within the order Lactobacillales (lactic acid bacteria), in the phylum Bacillota. Cell division in streptococci occurs ...
'', it plays an important role in immunology of infants in human milk feeding. Whereas the skin is a protective barrier due to its dryness and acidity, the
conjunctiva
The conjunctiva is a thin mucous membrane that lines the inside of the eyelids and covers the sclera (the white of the eye). It is composed of non-keratinized, stratified squamous epithelium with goblet cells, stratified columnar epithelium ...
(membrane covering the eye) is, instead, protected by secreted enzymes, mainly lysozyme and
defensin. However, when these protective barriers fail,
conjunctivitis
Conjunctivitis, also known as pink eye, is inflammation of the outermost layer of the white part of the eye and the inner surface of the eyelid. It makes the eye appear pink or reddish. Pain, burning, scratchiness, or itchiness may occur. The ...
results.
In certain cancers (especially myelomonocytic leukemia) excessive production of lysozyme by cancer cells can lead to toxic levels of lysozyme in the blood. High lysozyme blood levels can lead to kidney failure and low blood potassium, conditions that may improve or resolve with treatment of the primary malignancy.
Serum lysozyme is much less specific for diagnosis of sarcoidosis than serum angiotensin converting enzyme; however, since it is more sensitive, it is used as a marker of sarcoidosis disease activity and is suitable for disease monitoring in proven cases.
Chemical synthesis
The first chemical synthesis of a lysozyme protein was attempted by Prof. George W. Kenner and his group at the University of Liverpool in England.
This was finally achieved in 2007 by Thomas Durek in Steve Kent's lab at the University of Chicago who made a synthetic functional lysozyme molecule.
Other applications
Lysozyme crystals have been used to grow other functional materials for catalysis and biomedical applications. Lysozyme is a commonly used enzyme for lysing gram positive bacteria. Due to the unique function of lysozyme in which it can digest the cell wall and causes
osmotic shock
Osmotic shock or osmotic stress is physiologic dysfunction caused by a sudden change in the solute concentration around a cell, which causes a rapid change in the movement of water across its cell membrane. Under hypertonic conditions - conditions ...
(burst the cell by suddenly changing solute concentration around the cell and thus the
osmotic pressure), lysozyme is commonly used in lab setting to release proteins from bacterium
periplasm
The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the ''periplasmic space'' in gram-negative bacteria. Using cryo-electron microscopy it has been found that ...
while the inner membrane remains sealed as vesicles called the
spheroplast
A spheroplast (or sphaeroplast in British usage) is a microbial cell from which the cell wall has been almost completely removed, as by the action of penicillin or lysozyme. According to some definitions, the term is used to describe Gram-negative ...
.
For example, ''E. coli'' can be lysed using lysozyme to free the contents of the
periplasm
The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the ''periplasmic space'' in gram-negative bacteria. Using cryo-electron microscopy it has been found that ...
ic space. It is especially useful in lab setting for trying to collect the contents of the periplasm.
Lysozyme treatment is optimal at particular temperatures, pH ranges, and salt concentrations. Lysozyme activity increases with increasing temperatures, up to 60 degrees Celsius, with a pH range of 6.0-7.0. The salts present also affect lysozyme treatment, where some assert inhibitory effects, and others promote lysis via lysozyme treatment. Sodium chloride induces lysis, but at high concentrations, it is an active inhibitor of lysis. Similar observations have been seen with the use of potassium salts. Slight variations are present due to differences in bacterial strains. A consequence of the use of lysozyme in extracting recombinant proteins for
protein crystallization
Protein crystallization is the process of formation of a regular array of individual protein molecules stabilized by crystal contacts. If the crystal is sufficiently ordered, it will diffract. Some proteins naturally form crystalline arrays, li ...
is that the crystal may be contaminated with units of lysozyme, producing a physiologically irrelevant combination. In fact, some proteins simply cannot crystalize without such contamination.
Furthermore, lysozyme can serve as a tool in the expression of toxic recombinant proteins. Expressing recombinant proteins in BL21(DE3) strains is typically accomplished by the T7-RNA-polymerase. Via IPTG induction, the UV-5 repressor is inhibited, leading to the transcription of the T7-RNA-polymerase and thereby of the protein of interest. Nonetheless, a basal level of the T7-RNA-polymerase is observable even without induction. T7 lysozyme acts as an inhibitor of the T7-RNA-polymerase. Newly invented strains, containing a helper plasmid (pLysS), constitutively co-express low levels of T7 lysozyme, providing high stringency and consistent expression of the toxic recombinant protein.
History
The antibacterial property of hen egg white, due to the lysozyme it contains, was first observed by Laschtschenko in 1909.
The bacteria-killing activity of nasal mucus was demonstrated in 1922 by
Alexander Fleming, the discoverer of
penicillin, who coined the term lysozyme. Fleming reported, saying. "As this substance has properties akin to those of ferments I have called it a 'Lysozyme'."
Fleming went on to show that an enzymic substance was present in a wide variety of secretions and was capable of rapidly lysing (i.e. dissolving) different bacteria, particularly a yellow "coccus" that he studied".
Lysozyme was first crystallised by
Edward Abraham
Sir Edward Penley Abraham, (10 June 1913 – 8 May 1999) was an English biochemist instrumental in the development of the first antibiotics penicillin and cephalosporin.
Early life and education
Abraham was born on 10 June 1913 at 47 Sout ...
in 1937, enabling the three-dimensional structure of hen egg white lysozyme to be described by
David Chilton Phillips
David Chilton Phillips, Baron Phillips of Ellesmere, KBE, FRS (7 March 1924 – 23 February 1999) was a pioneering, British structural biologist and an influential figure in science and government.
Research
Phillips lead the team which determ ...
in 1965, when he obtained the first 2-
ångström
The angstromEntry "angstrom" in the Oxford online dictionary. Retrieved on 2019-03-02 from https://en.oxforddictionaries.com/definition/angstrom.Entry "angstrom" in the Merriam-Webster online dictionary. Retrieved on 2019-03-02 from https://www.m ...
(200
pm) resolution model via
X-ray crystallography
X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...
. The structure was publicly presented at a
Royal Institution lecture in 1965.
Lysozyme was the second protein structure and the first enzyme structure to be solved via X-ray diffraction methods, and the first enzyme to be fully sequenced that contains all twenty common amino acids.
As a result of Phillips' elucidation of the structure of lysozyme, it was also the first enzyme to have a detailed, specific mechanism suggested for its method of catalytic action.
This work led Phillips to provide an explanation for how
enzymes speed up a chemical reaction in terms of its physical structures. The original mechanism proposed by Phillips was more recently revised.
See also
*
Egg allergy
Egg allergy is an immune hypersensitivity to proteins found in chicken eggs, and possibly goose, duck, or turkey eggs. Symptoms can be either rapid or gradual in onset. The latter can take hours to days to appear. The former may include anaph ...
References
External links
*
Proteopedia.org HEW LysozymePDBe-KBprovides an overview of all the structure information available in the PDB for Human Lysozyme C.
PDBe-KBprovides an overview of all the structure information available in the PDB for Hen egg white Lysozyme C.
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EC 3.2.1
Immunology
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