Historically, residues (NAG) in (NAG-NAM)3hexasaccharides, which is

Historically,
lysozyme was discovered in 1922 by Alexander Fleming thanks to a
remarkable accident in his lab when some nasal drippings were dropped
in a petri dish with a bacterial culture. Fleming observed that the
cells were lysed by this substance and led to a research in which
lysozyme was identified as the main bacteriolytic element.

From a
biological point of view, lysozyme is a 129 aminoacid residues enzyme
(EC 3.2.1.17) classified as a hydrolase due to its biological
function: the catalytic hydrolysis of 1,4-beta-linkages between
N-acetylmuramic (NAM) and N-acetyl-D-glucosamine residues (NAG) in
(NAG-NAM)3hexasaccharides,
which is the “core” polysaccharide of many bacterial cell walls
(Figure 1), and between N-acetyl-D-glucosamine residues in
chitodextrins. The mechanical weakening of the rigid bacteria cell
wall renders bacterial susceptibility to osmotic lysis in hypotonic
media. Therefore, lysozyme has a defensive function against Gram
positive bacterial cells as it is present in mucosal secretions as
human milk, saliva or tears to destroy the peptidoglycan bacterial
cell wall. It has also been described that in bacteriophages lysozyme
is used as an agent to break the host bacterial cell and allow the
virus to inject its DNA and to lyse the bacteria after multiplication
for the releasing of new viruses1.

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Figure
1. The beta 1-4
glycosidic bonds between N-acetylglucosamine (NAG) and
N-acetylmuramic (NAM) to be hydrolysed by lysozyme are circled1.

The
globular structure of HEWL (Figure 2) contains five alpha helical
regions displayed in red, five beta sheets displayed in yellow and
some beta turns and large random coils displayed in green. Also,
another structural feature of lysozyme is the conformation of a beta
hairpin motif of supersecondary structure by the antiparallel
beta-pleated sheet, which is involved in the stability of the protein
by providing the correct alignment of hydrogen bonds.

Figure
2. Cartoon
representation of HEWL where different secondary structures are shown
in different colours. Alpha helix in red, beta strand in yellow and
turns/random in green.

Despite
lysozyme can hydrolyse different structurally substrates, the optimal
one is a (NAG-NAM)3hexasaccharide
which is cleaved at the NAM4-?-O-NAG5glycosidic
bond. Therefore, in the active site of lysozyme we can identify six
different residues involved in binding the six sugar rings of the
(NAG-NAM)3 hexasaccharide,
these being designated as rings “A” through “F”, and two
catalytic residues involved in the hydrolysis of the glycosidic bond
between rings “D” and “E”. The binding domains of HEWL are
TRP62, TRP63, ASN59, ALA107 and GLN57, while catalytic residues are
ASP52 and GLU35. The six binding residues form a deep cleft in the
protein surface into which the six rings of the hexapolysaccharide
can bind and be specifically hydrolysed into a disaccharide and a
tetrasaccharide2.
Therefore, the two catalytic residues are specifically located in
this deep cleft in order to preferentially cleave the glycosidic bond
connecting rings “D” and “E” (Figure 3). Smaller saccharides
are effective competitive inhibitors of lysozyme catalytic activity.
One example is the trisaccharide NAG3,
which specifically binds to the binding sites “A-C” and thus it
is positioned away from the two key catalytic residues (Figure 4).
However, the structure determination of lysozyme bound to this
inhibitor is a good approach to understand how (NAG-NAM)3
is embedded into the protein when lysozyme
breaks the glycosidic bonds in the bacterial cell walls3.

Figure
3. Cartoon
representation of HEWL where binding and catalytic residues are
labelled and highlight from the rest of the structure in red and
green respectively.

Figure
4. Surface
representation of HEWL and NAG3
lysozyme inhibitor bound to it. Binding residues in the catalytic
cleft are highlighted in red. Catalytic residues are highlighted in
blue.

The two
carboxylic acid residues ASP52 and GLU35 play an essential role in
catalysing the hydrolysis of the oligosaccharide substrate. The
hydrolysis of a glycoside bond corresponds to the conversion of
acetal to hemiacetal in which the protonation of the reactant oxygen
prior to bond cleavage is involved and further covalent resonance
stabilization of the transition state intermediate is required.
Therefore, the acid catalysis and covalent resonance stabilization is
adequately provided by both ASP52 and GLU35 acidic residues. The
reaction mechanism of lysozyme (Figure 5) can be divided into two
major steps: (I) hydrolysis of glycosidic bond by distortion of the
bond between NAM and NAG generating a glycosyl enzyme intermediate,
and (II) the reaction of a water molecule with the transition state
rendering the to different oligosaccharides and the unmodified
enzyme2.

Figure
5. Lysozyme mechanism
of action where the two major steps involving acidic residues ASP52
and GLU35 as well as a water molecule are shown2.

1.2Lysozyme
crystallization
In 1965, the first
structure of lysozyme was resolved
by X-ray analysis with 1 amstrong resolution by David Chilton
Philips. Since then, lysozyme has been a protein model for protein
crystallization and X-ray diffraction research and teaching due to
its many unique properties: (I) the ease to purify the
protein, (II) the ease to crystallise it
and (III) the very high resolution obtained from X-ray lysozyme
crystals diffraction. In this article we are going to work with Hen
Egg White Lysozyme (HEWL), which is an affordable model protein
extensively studied that can be easily crystallised.

Lysozyme has
been crystallised under many different conditions including
temperature, pH and precipitant agents. One of the most common
conditions for lysozyme crystallization is under low pH values
(4.5-5) and in the presence of different anions, which are
responsible for the solubility curve of the protein. In particular,
HEWL is one of the few examples in which chloride ions are used for
crystallization. Also, the combination of ammonium sulfate with
acetate buffer was demonstrated to be a good approach to obtain
crystals4.One of
the most successful approaches for lysozyme crystallization has been
the use of NaCl 5% (w/v) and acetate buffer with different pH values
ranging from 4 to 7. These experiments have yielded lysozyme
tetragonal crystals with a resolution of 1.75-2 amstrongs, which
demonstrate the effectivity of this method3.However,
further research has demonstrated the versatility of lysozyme as a
model protein for crystallization as it can be crystallised from
different sulfate salts, acidic and basic pH, in a wide range of
protein concentrations, at different temperatures and rendering
different crystal shapes (mainly tetragonal5,
orthorhombic6
and hexagonal 7)
which show different properties during crystal growth. Therefore,
lysozyme is widely used as a model for the study of crystallization
conditions and phenomena.