Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
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Peptidoglycan hydrolases as novel tool for anti-enterococcal therapy
Lenka Maliničová, Mária Piknová, Peter Pristaš, and Peter Javorský
Institute of Animal Physiology, Slovak Academy of Sciences, Šoltésovej 4-6, 04001 Košice, Slovakia
Enterococci are representatives of Gram-positive lactic acid bacteria. Most of them are constituents of commensal
microflora on human and animal mucous membranes, but some strains have emerged as a major cause of nosocomial
infections especially due to the presence of multi-drug resistance. It is anticipated that soon enterococcal infections will be
untreatable by current antibiotics, and alternative means of combating infections caused by these organisms will be
urgently needed. Finding new, more efficient therapeutics is a big challenge for pharmaceutical industry. One of the most
potential novel alternative to classic antibiotics are peptidoglycan hydrolases; enzymes degrading the major component of
bacterial cell wall. Thanks to their common features - bacteriolytic activity and narrow target spectrum, peptidoglycan
hydrolases represent a safe tool for management of diseases caused by antibiotic-resistant enterococcal strains.
Keywords peptidoglycan; hydrolase; Enterococcus
1. Introduction
Peptidoglycan hydrolases represent a broad group of enzymes of different origin, but similar structure. Their substrate;
peptidoglycan, is the most important component of bacterial cell walls. Activity of these enzymes results in a cell wall
disruption and consequent bacterial cell lysis. They participate in many physiological processes in a bacterial life cycle;
one class (endolysins) represent an important equipment of bacteriophage lytic arsenal, another is necessary in
processes involved in a peptidoglycan reassembly (autolysins) or a competitive battle among related bacterial strains
(bacteriocins).
Gram-positive bacteria are an appropriate target group for peptidoglycan hydrolases since in this case a
peptidoglycan layer serves as the first defense line of cell integrity. Within this group of bacteria we can find many
human and animal pathogens including some enterococcal strains. Infections caused by them become hard manageable
due to the increasing antibiotic resistance and therefore it is important to find some novel and efficient antibacterial
weapons. Peptidoglycan hydrolases seem to be a suitable alternative to antibiotics. An effectivity of some
representatives was tested and demonstrated in vitro and in vivo in animal models. We can find a growing number of
publications concerning characterization of these enzymes and their use in many different applications.
This paper is aimed at general characterization of peptidoglycan hydrolases, their features, classification,
physiological properties, and potential applications. Last section of the paper presents findings obtained at our institute.
2. Bacterial cell wall
One of the most important elements of bacterial cell is a cell wall, which is responsible for cell shape maintenance and
protecting against osmotic lysis. The strength and rigidity of the cell wall results from a layer of peptidoglycan, which is
a covalent macromolecular structure consisting of strong glycan chains that are crosslinked by flexible peptide bridges
[1] as shown in Fig. 1. The glycan strands are made up of alternating N-acetylglucosamine (NAG) and N-acetylmuramic
acid (NAM) residues linked by β-1→4 bonds. The D-lactoyl group of each NAM residue is substituted by a side
peptide chain whose composition is most often L-Ala-D-Glu-L-Lys-D-Ala [2]. Neighbouring glycan strands are
connected by crosslinking peptide chains between two side peptide chains. Crosslinking chains are the most variable
components of peptidoglycan, and their composition is species-specific.
Peptidoglycan layer has some unique biophysical properties; on the one hand, it has the strength to withstand the
cell’s turgor of up to 25 atmospheres, on the other hand it is not rigid, but flexible allowing reversible expansion of the
cell wall [2].
2.1 Gram-positive versus Gram-negative bacteria
Bacteria can be divided into two basic groups; Gram-positive and Gram-negative. Formerly, this differentiation was
based on the result of Gram staining, later it was uncovered that these different results were caused by diverse cell wall
composition. Figure 2 shows general differences between these two types of cell walls. In the case of Gram-negative
bacteria, there are three principal layers in the cell envelope; the inner (cytoplasmic) phospholipid membrane, the
peptidoglycan cell wall, and the outer membrane made of lipopolysaccharides, phospholipids, lipoproteins and porins.
In Gram-positive bacteria the outer membrane is absent but the peptidoglycan layer in their cell wall is many times
thicker than is found in Gram-negative bacteria [3]. Except peptidoglycan, Gram-positive cell wall contains one more
important component, teichoic acids. These anionic polymers occur in two distinct forms depending on whether they
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are linked to the peptidoglycan (teichoic acid) or to the head groups of cytoplasmic membrane lipids (lipoteichoic acid)
[4].
Fig. 1. Schematic representation of the peptidoglycan network structure. Long and strong glycan strains consisting of Nacetylglucosamine and N-acetylmuramic acid subunits are crosslinked by short and flexible peptide bridges.
Fig. 2.
Schematic structure of the cell envelope in a) Gram-negative, b) Gram-positive bacteria.
2.1.1
Enterococcal cell wall
Enterococcal species are common constituents of the intestinal flora of humans and animals. However, over the past
few decades, some strains have emerged as frequent causes of multiple antibiotic resistant nosocomial infections [5]. As
typical representatives of Gram-positive bacteria, enterococci have cell walls with very thick peptidoglycan layer closefitting the cytoplasmic membrane. Walls of enterococci represent 27 to 38% of the dry cell weight and contain three
main constituents; peptidoglycan, teichoic acid, polysaccharide, and sometimes, proteins are also mentioned. Variety
and number of non-peptidoglycan polymers is related to bacterial species and growth conditions.
One of the characteristic traits of enterococcal cell wall is the structure of crosslinking peptide chain in
peptidoglycan. The cross-bridge of most known Enterococcus species is formed by a singe D-Asp residue [6].
3. Peptidoglycan hydrolases
Bacterial cell wall hydrolases represent various and numerous group of enzymes, which are able to cleave
hydrolytically chemical bonds in peptidoglycan. Activity of these enzymes results in a cell wall disruption and
consequent bacterial cell lysis. In the last years, they have been studied extensively in many laboratories all around the
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world. The physiological functions of peptidoglycan hydrolases include the regulation of cell wall growth, the turnover
of peptidoglycan during growth, the separation of daughter cells during cell division and autolysis [7]. They are also
known to be produced by bacteriophages to digest the bacterial cell wall for phage progeny release at the end of
bacteriophage lytic cycle [8].
3.1
Common classification
Sources of peptidoglycan hydrolases are very variable. They include animals, insects, plants, bacteria, and phages.
Although enzymes from different sources may have similar structures and functions, they display important differences
with significant impacts on their clinical applications [9]. According to their sources, peptidoglycan hydrolases can be
classified into lysozymes, bacteriocins, autolysins, and endolysins.
3.1.1
Lysozymes
Lysozyme was discovered by Fleming in 1922. This bacteriolytic protein is phylogenetically ancient and almost
ubiquitous among living organisms. It is probably the most studied enzyme in biology, biochemistry and medicine [10].
Lysozymes sensu lato are widespread in nature. They are produced by bacteriophages, bacteria, fungi, invertebrates,
and vertebrates. In plants and animals (lysozymes sensu stricto), they represent a natural defense mechanism against
bacterial pathogens [11]. The most known and commonly used representative of this group is hen egg-white lysozyme.
3.1.2
Bacteriocins
As early as a half century ago, a relatively broad study concerning bacteriocins was published. In this work, bacteriocins
were characterized as a natural class of proteinaceous antibiotics, which in contrast to all other antibiotics, act only on
strains of the same or closely related species [12]. Bacteriocin production has been detected in all major lineages of
Eubacteria and in Archaebacteria. These highly specific enzymes are usually produced during stressful conditions and
can eliminate neighbouring cells that are not resistant to their effect. The primary role of bacteriocins is probably to
mediate intraspecific, or population-level, interactions [13]. Bacteriocins produced by Gram-positive lactic acid bacteria
are predominantly small (< 10 kDa) proteins, but several large bacteriocins with cell wall hydrolytic activity have been
described.
3.1.3
Autolysins
Autolysins represent a group of peptidoglycan hydrolases that digest the cell wall of bacteria that produce them. They
are involved in numerous cellular physiological processes including cell growth, cell-wall turnover, peptidoglycan
maturation, cell division, separation, differentiation, etc [14].
3.1.4
Endolysins
Bacteriophage endolysins are enzymes that are expressed near the end of the phage lytic cycle. Subsequent host cells
lysis allows the bacteriophage progeny to escape from the host cell (“lysis from within”). The purified endolysins can
also be exposed to bacteria externally, causing “lysis from without” [15].
3.2
Substrate specificity of peptidoglycan hydrolases
According to the specific bond in the peptidoglycan that is split down, peptidoglycan hydrolases can be divided into
several groups, as shown in Fig. 3. Glycosidases (N-acetylglucosaminidases and N-acetylmuramidases) hydrolyze β1→4 bonds between disaccharide units of glycan chains, peptidases are capable of cleaving short peptides
(carboxypeptidases) or peptide cross-bridges (endopeptidases), and N-acetylmuramoyl-L-alanine amidases (amidases)
cleave the amide bond between glycan chains and peptide bridges. Cleavage specificity depends on the catalytic domain
of a peptidoglycan hydrolase [2, 16, 17] (see below).
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Fig. 3. Substrate specificity of peptidoglycan hydrolases.
3.3
Domain structure of peptidoglycan hydrolases
Molecule of typical peptidoglycan hydrolase consists of two domains, as shown in Fig. 4. With some exceptions, the Nterminal domain contains the catalytic activity (glycosidase, peptidase, or amidase) and is responsible for cleaving
chemical bonds in the substrate – peptidoglycan. In some cases, two and perhaps even three different catalytic domains
may occur in one enzyme molecule.
Fig. 4. Schematic structure of typical peptidoglycan hydrolase.
This domain (or modular) structure of peptidoglycan hydrolases offers a possibility to construct new chimeric
enzymes. The DNA sequences coding for the modules of different origin can be exchanged allowing the resulting
chimeric proteins to display the biochemical properties of the modules that constitute the hybrid enzyme [18].
3.3.1
Binding receptors for CWB domains
C-terminal domain (called cell wall targeting – CWT, or cell wall binding – CWB domain) usually targets whole
enzyme to the bacterial cell wall [8]. The activity level of peptidoglycan hydrolases does not arise only from catalytic
domains. Efficient cleavage requires binding of CWB domain to its cell wall substrate. CWB domains do not recognize
the peptidoglycan itself, since its structure is highly conserved. Correctly binding of whole enzyme molecule to the cell
wall of sensitive bacteria requires certain specific receptors in peptidoglycan layer. This targeting ability offers the
enzymes a high degree of specificity, since these receptors are only found in enzyme-sensitive bacteria [8, 19].
Receptors for some peptidoglycan hydrolases have been known for a long time, the nature of others is still being
investigated. One of the formerly discovered is the receptor for CWB domain of pneumococcal autolysin (Nacetylmuramoyl-L-alanine amidase). Pneumococci have a nutritional requirement for choline and this amino alcohol
seems to be incorporated exclusively into the teichoic acids of this bacterium. Replacement of choline in the growth
medium with ethanolamine results in the formation of ethanolamine-containing cell walls, which were totally resistant
against the action of the pneumococcal autolysin. Based on this finding, choline has been considered as a receptor for
CWB domain of this enzyme [20].
Lysostaphin – bacteriocin from Staphylococcus simulans bv. staphylolyticus and its homologue, hydrolase ALE-1
from S. capitis specifically recognizes cell walls of S. aureus. These glycylglycine endopeptidases cleave the peptide
cross-bridges in the peptidoglycan [21]. The CWB domain of these hydrolases has been classified as an SH3 domain
and it has been uncovered that they recognize pentaglycine cross bridges in peptidoglycan, which are characteristic just
for S. aureus [22].
On the other hand, there are some CWB domains with much wider range of receptors. For example, LysM (Lysin
Motif) domain, which has been found in more than 4000 proteins of both prokaryotes and eukaryotes, represents the
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main peptidoglycan-binding domain in bacteria. This domain binds to various types of peptidoglycan and most likely it
recognizes the N-acetylglucosamine moiety [23].
3.4
Applications of peptidoglycan-degrading enzymes
Enzymes have been selected by natural evolution to favour the development of living organisms in their natural
environment. Consequently, not all natural enzymes have adequate properties (specificity, stability, catalytic activity) to
make them suitable for biotechnological applications, thus there are required several strategies to modify their natural
properties [24].
Peptidoglycan-degrading enzymes have greatly been used in biotechnology industry to break cells. Major
applications of these enzymes are related to the extraction of intracellular substances from bacterial cells and preparing
spheroplasts for cell transformation. Other applications are based on the antimicrobial properties of these enzymes [17].
3.4.1
Obtaining intracellular products
Peptidoglycan hydrolases (principally lysozyme) are suitable tools for bacterial cell wall degradation in methods and
techniques used for isolation of nucleic acids or releasing recombinant proteins from host bacterial cells. When
intracellular products are obtained from Gram-positive bacteria, the action of lysozyme is sufficient [25]. In Gramnegative bacteria, the outer membrane prohibits access to lytic enzymes, thus in this case pre-treatment with a detergent
or a cation chelating agent (as EDTA) is usually necessary to remove the outer membrane of Gram-negative cells [17].
3.4.2
Alternative to classic antibiotics
Enterococci have long been thought of as a harmless commensal of the mammalian gastrointestinal tract. However, in
the last two decades they have become an important cause of nosocomial infections which are often difficult to treat due
to the resistance to a large number of antibiotics, and an emerging resistance even to novel antibiotics used in the
therapy is often reported in literature [26, 27].
One of the most potential novel alternatives to classic antibiotics are peptidoglycan hydrolases thank to their common
features - bacteriolytic activity and narrow target spectrum. Several forms of these enzymes may be used in different
applications, e.g. purified native enzymes, modified natural forms [28], recombinant enzymes prepared in a host
organism and applied externally [29], or even overexpressed endogenously in transgenic plant or animal organisms to
enhance their resistance against bacterial diseases [30].
The problem with infections caused by multidrug resistant bacteria also recalls the scientific interest in phage
therapy, which has been relegated during the antibiotic era. Due to some potential risks in the application of whole
phage particles (e.g. immunity response), purified phage endolysins seem to be a more suitable candidate.
3.4.3 Plant protection against phytopathogenic bacteria
The continuously increasing antibiotic resistance does not concern only human and animal pathogens but also
phytopathogenic bacterial species. It is also clear that antibiotic resistance in plant bacterial pathogens can be
transferred to humans via the food chain. In addition to the interest in using phage therapy (or purified phage
endolysins) in human medicine, there is much interest in the application of phages as a biocontrol tool for bacterial
species of agricultural importance [31].
3.4.4
Biocontrol of bacteria in food and feed
The empirical use of microorganisms and/or their natural products for the preservation of foods (biopreservation) has
been a common practice for millennia. A fitting example is the group of lactic acid bacteria involved in fermentation of
various kinds of food. A lot of studies have revealed that lactic acid bacteria produce an array of antimicrobial
substances, including bacteriocins [32].
At present, the most commonly and commercially available bacteriocin is nisin but the bio-preservating effect of
several more bacteriocins or a combination of nisin and lysozyme has been detected [32, 33]. Another promising way of
food biopreservation again seems to be an application of bacteriophages or their endolysins, since they are capable to
decontaminate raw products, such as fresh fruit and vegetables, to disinfect equipment and contact surfaces and to
extend the shelf life of perishable manufactured foods [34].
3.4.5
Cosmetics and pharmaceutical products
Thanks to their antimicrobial properties, peptidoglycan hydrolases play an undeniably important role in production of
cosmetic and pharmaceutical preparations. One of suitable candidates is again lysozyme, since its efficacy in therapy of
infections of the skin and mucous membranes has been confirmed. Several years (or even decades) ago it was shown
that lysozyme as an ingredient of ointments or poultices, accelerates the healing rate of burns and open skin wounds
[35, 36]. At the last time bacteriophage endolysins were investigated as therapeutic agents for bacterial skin infections
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in animals. Aerosolized PlyC endolysin was able to eradicate or significantly reduce pathogenic Streptococcus equi and
was proposed as the first protein-based, narrow-spectrum disinfectant against this bacterial strain [37].
4. Known peptidoglycan hydrolases with anti-enterococcal activity
Enterococci are a leading cause of nosocomial infections, and treating these infections with conventional antibiotics has
become increasingly difficult due to the acquisition of antibiotic resistance genes by these organisms [38]. Although
several peptidoglycan hydrolases of diverse origin with activity against Gram-positive bacteria were identified and
purified (e.g. Acd from Clostridium difficile [39], LytA from Streptococcus pneumoniae [40] or PL-1 from
Lactobacillus casei phage [41] etc.), currently only two enzymes with anti-enterococcal activity are known - endolysin
PlyV12 and enterolysin A.
4.1
Endolysin PlyV12
In 2004, PlyV12, a bacteriophage endolysin, was isolated and shown to effectively kill some enterococcal strains, as
well as other human pathogens. PlyV12 is an amidase that exhibits a lytic effect on multiple E. faecalis (also
vancomycin-resistant) strains. In contrast to other reported endolysins, PlyV12 was also found to be active against
several pathogenic streptococcal and staphylococcal strains, making it one of the first endolysins with a spectrum of
activity outside that of the host and closely related bacterial strains. This broad activity spectrum suggests the presence
of a unique cell wall carbohydrate receptor common to these different human pathogens [38].
The molecule of PlyV12 consists of catalytic Amidase_5 domain and cell wall binding SH3_5 domain, as shown in
Fig. 5.
Fig. 5. Schematic domain structure of endolysin PlyV12 obtained by the blastp analysis of its protein sequence using
http://blast.ncbi.nlm.nih.gov/Blast.cgi.
4.2
Enterolysin A
Enterolysin A (EnlA), firstly described in 2003, is a heat-labile bacteriocin produced by Enterococcus faecalis LMG
2333 strain, with a broad inhibitory spectrum including selected enterococci, streptococci, pediococci, lactococci, and
lactobacilli. Afterwards, an enterolysin A homologue was found to be produced by Gram-positive ruminal cocci and
enterolysin A structural gene homologues were identified in several enterococcal and streptococcal bacterial species
including Streptococcus bovis and Enterococcus malodoratus strains. Enterolysin A belongs to group of peptidoglycan
hydrolases, with typical modular structure. The N-terminal region of the protein contains a domain found in the M37
family of metallopeptidases (catalytic domain). The C-terminal part of EnlA is distantly related to the known hydrolases
only, but it shows significant sequence identity to two bacteriophage endolysins [42, 43]. This part has been thought to
be responsible for enterolysin A binding to its cell wall substrate (putative CWB domain).
The heterologous expression of enterolysin A was studied in Escherichia coli. The expression of EnlA structural
gene led to the synthesis and secretion of functional-active His-tagged enterolysin A protein (as shown in Fig. 6), which
was purified to homogeneity and was shown to be fully active against the indicator strain. The expression of N-terminal
or C-terminal part of EnlA and deletion of last 58 amino acids from C-terminal domain of EnlA led to the synthesis of
biologically non-active proteins [44]. The activity of EnlA relies upon cell wall binding and C-terminal truncation of the
protein completely abolishes its activity.
Fig. 6. Zone of growth inhibition caused by peptidoglycan hydrolase enterolysin A applied on sensitive Enterococcus malodoratus
strain. Result of agar diffusion test.
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In the following experiments, the putative function of enterolysin A C-terminal domain in cell wall targeting and
binding was demonstrated. C-terminal domain was fused to GFP reporter and binding experiments using whole cells of
various bacterial species as the target were performed. Figure 7 shows binding of fusion protein (named GFP-CWB) to
bacterial cell walls, as visualized by fluorescence microscopy.
Fig. 7. Decoration of the surface of E. malodoratus NCDO846 after incubation with GFP-tagged C-terminal domain of EnlA
hydrolase. Visualized by fluorescence microscopy.
Data obtained using fluorescence binding assay correlated with sensitivity/resistance of bacterial species to EnlA
hydrolase determined by agar diffusion test. As expected, GFP-CWB bound to the cells of bacteria classified as EnlA
sensitive according to agar diffusion test, causing fluorescence of their surfaces, and did not bind to the surfaces of
EnlA producing strain, or any bacterial species resistant to EnlA hydrolase. We demonstrated that the C-terminal region
of enterolysin A is involved in specific recognition and binding to the target cell envelopes and represents true cell wall
targeting domain.
5. Screening genomes of enterococci for genes encoding endolysins and endolysin-like
proteins
Aside from enterolysin A, only three other bacteriolytic bacteriocins produced by Gram-positive lactic acid bacteria
were described, namely zoocin A [45], stellalysin [46], and millericin B [47] and phages are probably the best source of
anti-enterococcal endolysins. Although the number of phages infecting enterococci is sufficient, only few of them have
been characterized at molecular level and the only known phage endolysin with activity against Enterococcus species is
PlyV12. Much higher numbers of phage sequences were passively acquired as prophages in Enterococcus spp. genome
sequencing projects. Therefore, we also analysed enterococcal genomes available in GenBank database for the
prophage sequences. We found 62 protein sequences of endolysin-like enzymes. All obtained sequences were
subsequently compared using BLASTP.
All of these endolysins appear to have a modular design (as described above). As shown in Table 1, these enzymes,
with some exceptions, possess muramidase-like activity combined with LysM binding domain, or amidase-like activity
combined with SH3_5 domain. Surprisingly, the Amidase_5 domain present in PlyV12 endolysin is rarely observed in
endolysin-like proteins from enterococcal genomes, despite it is frequently encountered in endolysins from other
members of Lactobacillales order (data not shown). The domain structure of bacteriophage endolysins represents a
potential for construction of novel recombinant proteins with artificial combination of individual domains (so-called
„domain shuffling“).
Table 1. Basic characteristics of selected endolysin-like proteins from enterococcal genomes (data were obtained in march
2010)
Access code
Description
Source
Catalytic domain
CWB domain
ZP_05592581
ZP_05593964
ZP_05592904
ZP_04438395
ZP_04438946
ZP_04437810
ZP_05474738
ZP_05475717
lysin
lysin
endolysin
endolysin
endolysin
endolysin
lysin
endolysin
E. faecalis AR01/DG
E. faecalis AR01/DG
E. faecalis AR01/DG
E. faecalis ATCC 29200
E. faecalis ATCC 29200
E. faecalis ATCC 29200
E. faecalis ATCC 4200
E. faecalis ATCC 4200
Amidase_2
GH25_muramidase
Amidase_2
GH25_muramidase
Amidase_2
GH25_muramidase
Amidase_2
Amidase_2
unknown
LysM
SH3_5
LysM
SH3_5
LysM
unknown
SH3_5
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ZP_05476312
ZP_05585395
ZP_05584633
ZP_05562195
ZP_05563475
ZP_05562950
ZP_05561234
ZP_05560950
ZP_05581557
ZP_05576004
ZP_05575058
ZP_05579809
ZP_05579618
ZP_03985506
ZP_03983336
ZP_05568908
ZP_05566663
ZP_05568662
phage lysin
endolysin
endolysin
lysin
lysin
E. faecalis ATCC 4200
E. faecalis CH188
E. faecalis CH188
E. faecalis DS5
E. faecalis DS5
E. faecalis DS5
E. faecalis DS5
E. faecalis DS5
E. faecalis D6
E. faecalis E1Sol
E. faecalis E1Sol
E. faecalis Fly1
E. faecalis Fly1
E. faecalis HH22
E. faecalis HH22
E. faecalis HIP11704
E. faecalis HIP11704
E. faecalis HIP11704
endolysin
endolysin
endolysin
lysin
lysin
lysin
endolysin
endolysin
endolysin
endolysin
phage lysin
lysin
lysin
endolysin
ZP_05567493 domainE. faecalis HIP11704
containing protein
AAT01859
endolysin PlyV12 Enterococcus sp. phage 1
GH25_muramidase
GH25_muramidase
GH25_muramidase
GH25_muramidase
GH25_muramidase
Amidase_2
Amidase_2
Amidase_2
GH25_muramidase
GH25_muramidase
Amidase_2
Amidase_2
Amidase_2
GH25_muramidase
unknown
GH25_muramidase
Amidase_2
GH25_muramidase
LysM
LysM
LysM
LysM
LysM
SH3_5
SH3_5
SH3_5
LysM
LysM
unknown
SH3_5
SH3_5
LysM
SH3_5
LysM
unknown
LysM
Amidase_5
SH3_5
Amidase_5
SH3_5
As a next step, a phylogenetic analysis of the catalytic and CWB domain sequences, respectively, using MEGA4
software [48] was performed (results not shown). The results obtained indicate that the degree of cognation among
catalytic domains in the most cases corresponds in most cases with degree of cognation among CWB domains of
individual proteins. One may hypothesize that at least in the case of peptidoglycan hydrolases the natural trend is
distribution of complete genes (as well-tried domain combinations) rather than separate domains. Despite that, an
artificial domain shuffling could be supposed as an indispensable method in enzymatic engineering.
6. Conclusions
Peptidoglycan hydrolases seem to be an interesting approach in the fight against multidrug resistant bacterial strains and
in the future they could be an inestimable tool for treating diseases caused by these microorganisms. Purification of
these proteins from their natural sources is often difficult and therefore preparation of recombinant hydrolases expressed
in the new host organisms appears the more appropriate way to obtain them. Moreover, domain structure of
peptidoglycan hydrolases offers some potential for enzyme engineering, since the combination of catalytic and CWB
domains of different origin could lead in novel enzyme molecules with many desirable features. However, common use
of these enzymes will require more studies and finding answers to many more research questions.
Acknowledgments The support by APVV Grant No. 0586-07 and VEGA Grant No. 2/0006/08 is gratefully
acknowledged.
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