Malaysian Journal of Biochemistry and Molecular
Biology (2008) 16(1), 1-10 1
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Review
Article
Snake Venom L-Amino Acid Oxidases and Their Potential
Biomedical Applications
Nget-Hong Tan and Shin-Yee Fung
Department
of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur,
Malaysia
Author
for correspondence: Prof. Dr. Nget-Hong Tan,
Department
of Molecular Medicine, Faculty of Medicine,
University
of Malaya, 50603 Kuala Lumpur. Tel: 603-79674912
Fax:
603-79674957 E-mail: tanngethong@yahoo.com.sg
Abstract
L-amino acid oxidase (LAAO) occurs widely in snake
venoms. The enzyme is highly specific for L-amino acids, and generally hydrophobic
amino acids are the best substrates. LAAO is a flavoprotein consisting of two
identical subunits, each with a molecular mass of approximately 60 kDa. The
purified enzymes are glycoproteins with 3-4% carbohydrate. Deglycosylation of
the enzyme did not alter the enzymatic activity but appeared to alter its
pharmacological activities. The amino acid sequences of snake venom LAAOs
showed a high degree of homology. X-ray structural analysis of LAAO revealed a
dynamic active site and the presence of 3 domains: a FAD-binding domain, a
substrate-binding domain and a helical domain. LAAOs were reported to exhibit
moderate lethal toxicity. Recent studies showed that LAAOs are multifunctional
enzymes exhibiting edema-inducing, platelet aggregation inducing or inhibiting,
apoptotic inducing as well as
anti-bacterial, anti-coagulant and anti-HIV effects. These effects are
mostly mediated by the H2O2 liberated in the oxidation process but direct
interactions between LAAO and the target cells may play an important role.
High resolution X-ray structure of the enzyme revealed
the presence of a channel that would direct the H2O2 product to the exterior surface
of the protein, near the glycan moiety at Asn 172. The glycan moiety was
thought to be involved with LAAO-target cell interaction. This may explain the
ability of LAAO to localize H2O2 to the targeted cells. A better understanding
of the pharmacological actions of LAAOs will facilitate the application of
snake venom LAAOs in the design of anti-cancer and anti-HIV drugs as well as
drugs for the treatment of infectious diseases caused by parasites such as
leishmaniasis.
Keywords: L-amino
acid oxidase, snake venom, Calloselasma rhodostoma (Malayan pit viper).
Introduction
L-Amino acid oxidase (L-amino acid:O2 oxidoreductase, E.C.
1.4.3.2.) is a flavoenzyme that
catalyzes the oxidative deamination of an L-amino acid to form the
corresponding α-ketoacid and ammonia:
RCH(NH3+)COO- + O2 + H2O + RCOCOO- + NH4 + + H2O2
L-Amino acid oxidase
(LAAO) occurs widely in nature [1] and snake venoms are perhaps the richest
sources of the enzyme. Snake venom LAAOs are generally very active and have
been used widely in preparation of α-keto acids because of their chemo- and
stereospecificity
[2, 3]. α-Keto
acids of essential amino acids are useful nutraceuticals as well as therapeutic
agents for certain diseases.
Recently, snake venom LAAO has become an interesting object for biomedical studies
because of its antimicrobial, anti-HIV, anticoagulant, platelet aggregation-inducing
and inhibiting, apoptotic-inducing as well as anti-cancer activities.
Snake venom LAAO is recognized as a multifunctional
protein with promising biomedical
application. Several reviews on snake venom L-amino acid oxidases have been
published [1, 4-9].
LAAO Assay Methods
Many methods of LAAO assay are available [1]. The O2
electrode technique has been widely used, particularly in kinetic studies. A commonly
used spectrophotometric method was described by Bergmeyer, which measured the rate of oxidation by measuring the rate
of formation of color complex between the hydrogen peroxide produced and
o-dianisidine [10]. Based on the same principle, a spectrophotometric
microplate assay has been developed suitable for processing large numbers of
samples [11].
LAAO Occurrence in Snake Venoms
LAAO can be found in venoms from most genera [12]. The richest
sources of LAAO are the crotalid venoms. The
enzyme usually constitutes 1-4% of the venom by weight but in Calloselasma
rhodostoma (Malayan pit viper) it constitutes up to 30% by weight of the
dried venom [8]. Venoms from mamba and sea snakes either
contain no or trace amount of L-amino acid oxidase activity.
Purification of Snake Venom LAAOs
Since 1990s, many authors have reported the purification
and characterization of LAAOs from various snake venoms (Table 1). In some
snake venoms, the enzymes present were in many isoforms. Hayes and Wellner, for
example, reported that there were at least 18
isoforms of the LAAO in Crotalus adamanteus venom, and that
glycosylation contributes to the microheterogeneity Snake venom L-amino acid
oxidases 2 for the enzyme [13]. However, microheterogeneity was not
observed for LAAOs isolated from most other venoms. In general, it is
relatively easy to obtain homogenous LAAO from snake venom. For example, the
LAAO from C. rhodostoma venom can be obtained using a simple two-step
procedure: Sephadex gel-filtration chromatography followed by Mono-Q high
performance ion exchange chromatography (Figure 1) [14].
Physical Properties of Snake Venom LAAOs
General physical properties
Snake venom LAAOs generally have molecular mass ranging
from 112 kDa to 140 kDa as determined by gel filtration chromatography and
57-68 kDa by SDSpolyacrylamide gel electrophoresis, indicating that the enzymes
are dimers and usually with identical subunits (Table 1). Snake venom LAAOs
have a wide range of isoelectric points, ranging from 4.4 to 8.12 [8]. LAAO is a
flavoprotein with two molecules of flavin coenzymes. The flavins, which exhibit absorption maximum
at 275, 390 and 462 nm, are responsible for the yellowish color of the enzyme
as well as for the venoms. Most authors reported that the flavin coenzymes are
both FAD though some earlier reports suggested FMN as the coenzymes [8].
Snake venom LAAOs are stable at room temperature and at 4°C. Ophiophagus hannah LAAO,
for example, at pH 7.4, retained 100% and 80%, respectively, of activity after
incubating at 37°C for 5, and 14 days. Many LAAOs, however, are unstable at alkaline condition. Some
snake venom LAAOs have highly sensitive active sites. For example, C.
adamanteus LAAO undergoes reversible pH or temperature-dependent
inactivation, accompanied by structural changes in flavin binding site though
retaining its overall secondary structure. Earlier, Curti et al. reported
that C. adamanteus LAAO was inactivated by storage at -5°C and -60°C,
and by freeze-drying. Many other snake venom LAAOs are also inactivated by freezing. Generally, the
inactivated enzyme can be reactivated
completely by heating at pH 5. The inactivation was accompanied by shifts
in absorption spectrum and optical rotary dispersion Snake venom L-amino
acid oxidases 3 spectrum, and reactivation reverses the spectra changes completely. The inactivation was
believed to be due to a limited conformational change of the enzyme structure, presumably
also in the vicinity of the flavin biding site. This has been substantiated by
X-ray structural studies. Some snake venom LAAOs (for example, LAAOs from O.
hannah and C. rhodostoma), however, are not inactivated by freezing.
Reconstitution
of LAAO
As a result of the high sensitivity of many snake venom
LAAOs to their microenvironment, it was not possible to prepare reconstitutable
apoprotein, as reconstitution with the FAD coenzyme often resulted in an
inactive protein, with a perturbed conformation of the flavin binding site.
Raibekas and Massey reported near complete activation of the reconstituted
apoprotein and the restoration of its native flavin binding site in the presence
of 50% glycerol [18]. Glycerol as a co-solvent plays a special role in this
restorative process by induction of rearrangement in the protein structure. The
authors suggested that hydrophobic effect appears to be the dominating force in
this in vitro-assisted restorative process.
Structure : Snake venom L-amino acid oxidases 5
The authors suggested that one portion of this channel
may serve as the entry path for O2 during the oxidative half-reaction. On the
other hand, the second region, which was separated from the proposed O2 channel
by the N terminus (residues 8-16) of the protein, may play a role in H2O2
release. Presumably, the channel would direct the H2O2 product to the exterior
surface of the protein, near the glycan moiety at Asn 172, which was thought to
anchor the enzyme to the host cell. This channel location may explain the
ability of the enzyme to localize H2O2 to the targeted cell and thus induce the
apoptotic effect as well as other pharmacological activities. The X-ray
structure confirmed that the carbohydrate moieties are linked to Asn 172 and
Asn 361. The authors speculated that the disialylated oligosaccarides at Asn 172,
which is located in the vicinity to the channel leading to the active site of
the enzyme, may bind to siglecs (sialic acid-binding Ig superfamily lectins) of
the target cells via its sialylated glycan moiety, and may then result in
production of locally high concentration of H2O2 in or near the binding
interface. This, in turn, could lead to oxidative damage of the siglec or
another adjacent cell structural element.
Enzymatic Properties of LAAOs
General enzymatic properties
LAAO required Mg2+ and was inhibited by Ca2+, phosphate
as well as p-chloromercuribenzoate. Certain amino acids stabilize the enzyme,
while at highn concentration they become inhibitors. The enzyme is also
competitively inhibited by various aliphatic and aromatic acids and had a pH
optimum of between 7 and 8.5 [8]. LAAO from different sources differ
substantially in their specific activity. When L-leucine was used as the
substrate, at pH 8.5, the specific activities of the enzymes isolated from C.
rhodostoma, N. kaouthia and O. hannah were 0.54, 4.59 and 20.9 μmole/min/mg,
respectively. Substrate inhibition occurs at high substrate
concentrations.
Over the last 15 years, LAAOs have become an interesting
object for biomedical studies because of its apoptotic, cytotoxic, platelet aggregation, anticoagulant and other
physiological effects. These effects are thought to be mediated by the
chemically very reactive hydrogen peroxide
generated in the oxidation process, because H2O2 scavenger such as catalase
neutralizes the effects. Sometimes the toxic effects cannot be attributed to
H2O2 liberated alone and direct interactions between LAAO and the target cells
may play an important role [23].
Edema-inducing and hemorrhagic activities
Several authors reported that venom LAAO was able to
induce extensive edema in the mouse paw, and some with slight hemorrhages [9,
28, 29, 40]. Tan and Choy reported that O. hannah LAAO exhibited strong
edema inducing activity [41], and the enzyme elicited a ‘delayedtype’ time
course of edema formation, indicating that the edema formation caused by LAAO
was not mediated through release of amines subsequent to mast cell degradation,
which usually elicited a ‘rapid’ type of edema formation. The edema-inducing
activity of the enzyme was not inhibited by diphenhydramine or dexamethasone. Izidoro
et al. suggested that edema formation is due to activation of the
inflammatory response by the H2O2 generated, as administration of glutathione
to the mouse paw inhibited the edema-inducing activity of the enzyme [29]. The
hemorrhagic effect of LAAO results from complex effects, and may involve
apoptosis of endothelial and other vascular cells.
It is still not clear why some LAAOs induce and others
inhibit platelet aggregation. Sakurai et al. suggested that the
controversies may be connected with differences in the experimental procedure
or preparation of blood samples. Other possibilities include the difference in
specific activity of the enzyme, or the involvement of mechanisms other than
H2O2 liberation that are present only in certain LAAOs.
Apoptosis-inducing
effect
Apoptosis is the programmed cell death characterized by
a distinct pattern of cellular events, including cleavage of nuclear DNA into
fragments that produce a typical nucleosomal DNA ladder in agarose gel. Snake
venom is known to exhibit apoptosis-inducing effect. Suhr and Kim [47] and
Torii et al. [48] reported that the snake venom component that induced
apoptosis was an LAAO, and that the LAAO induced apoptosis in human umbilical vein
endothelial, human promyelocytic
leukemia HL-60, human ovarian carcinoma A2789 and mouse endothelial KN-3
cells. Since then, many snake venom LAAOs were reported to also exhibit
apoptosis-inducing activity [22, 29, 32-33, 49-50], and the apoptosis was
usually demonstrated by the DNA fragmentation gel pattern. The apoptosis-inducing
activity was abolished by catalase and other H2O2 scavengers, indicating that
the H2O2 generated by LAAO action plays
an important role in the apoptosis.
Tempone et al. suggested that cells submitted
to oxidative stress induced by LAAO generated H2O2 that could activate heat
shock proteins and initiate cell membrane disorganization, DNA fragmentation,
apoptosis and therefore cell death [51]. Sun et al. suggested that the generated
peroxide activates the transcription of such factors as the nuclear factor B,
the activator protein 1, Fas/Apo-1 and p53 [49].
Suhr and Kim, however, demonstrated that LAAO induced apoptotic
mechanism was clearly distinguishable from
the one stimulated directly by exogenous H2O2, suggesting that the LAAO-induced
apoptosis was not solely triggered by the peroxide produced by the oxidation [27].
Takatsuka et al. demonstrated that venom LAAOs directly
bind to cell surface thereby increasing the local peroxide concentration [22].
On the other hand, Torii et al. reported that the venom LAAO did not
associate with human embryonic kidney cells [20]. The cause of these
discrepancies is not clear.
Ande et al. [50] and Samel et al. [33],
using Jurkat and K562 (human chronic
myeloid leukemia) cells, respectively, reported that at low concentration LAAO induced apoptosis, but caused necrosis of the
cells at higher concentrations. According to Ande et al. the factors
contributing to apoptosis are: (i) generation of toxic
intermediates from fetal calf serum and (ii) binding and internalization of
LAAO, which appears to be mediated by the glycan moiety of the enzyme, as
desialylation of the enzyme reduces cytotoxicity [50]. D-Amino acid oxidase,
which lacks glycosylation, also triggers necrosis by the H2O2 liberated, but it
does not cause apoptosis. Thus, just like its effect on platelet aggregation,
induction of cell death by LAAO also appears to involve both the generation of
H2O2 and the molecular interaction of the
8 glycan moiety of the enzyme with structures at the
cell surface.
Antibacterial
activity
Stiles et al. reported that two LAAOs from the
venom of Pseudechis australis (Mulga snake) have a powerful antibacterial
effect against Gram-positive and Gramnegative bacteria [52]. Compared to
tetracycline, the in vitro antibacterial effects of the enzymes were
18-70
times more effective, on a molar basis. Recently, many
authors reported LAAO from other snake venoms also exhibited similar
antibacterial activity [28-29, 34, 45]. It is believed that the antibacterial
effect of LAAO is also due to the H2O2 liberated, as addition of catalase completely
suppressed the antibacterial activity. Electron microscopic studies suggested
that the H2O2 generated in the oxidation process induced bacterial membrane
rupture and then cell death [45]. Zhang et al. reported that the A. halys
LAAO was able to bind to the surfaces of bacteria and generate high
concentrations of H2O2 locally, which enables the enzyme to inhibit bacterial
growth at low concentrations [53]. It is not clear whether this happens to
other snake venom LAAOs.
Leishmanicidal
activity
Leishmaniasis includes a spectrum of human infectious disease
ranging from self-healing cutaneous ulceration to a progressive and lethal
visceral infection. It is a disease that probably affects 12 million people and
is prevalent in 88 nations throughout the world. Tempone et al. [51]
and Toyama et al. [45] reported that snake
venom LAAO possesses strong leishmanicidal activity, as the H2O2 generated by
the enzyme was a strong inducer of apoptosis in promastigotes of Leishmania
ssp. cells. At present, few drugs are available for treatment of
leishmaniasis.
The understanding of the mode of action of LAAO upon parasites
may trigger the design of new drugs or therapeutical approaches for
leishmaniasis. For example, if one was able to target a H2O2 generator, (such
as snake venom LAAO) towards the parasitophorous vacuole, this
would
represent a highly specific treatment not only for leishmaniasis but also for
other intracellular parasites.
Anti-HIV
activity
Zhang et al. reported that LAAO isolated from T.
stejnegeri venom possesses antiviral activity [23]. The enzyme exhibited
dose-dependent inhibition on HIV-I infection and replication at concentrations
that showed little effect on cell viability. Under the same experimental conditions,
no anti-HIV-1 activity was observed by exogeneous addition of H2O2.
Furthermore, the presence of catalase causes a decrease in its antivirus
activity but resulted in an increase of its antiviral selectivity. The authors
suggested that while liberated H2O2 is involved in the anti-HIV-1 activity of
the LAAO, the dosages of H2O2 and relative molecular pathways mediating suppression
in virus infection and replication are independent and/or different from those
of causing cell death.
Presumably, the mechanism of the anti-HIV-1 effect of
LAAO involves specific binding of the enzyme to cell membrane, which helps to
generate high local concentrations of H2O2 to trigger certain signal reactions and
activation of host cells, resulting in the inhibition of HIV infection and/or
replication.
Conclusion
Prior to the 1990s,
studies of snake venom LAAO dealt mainly with their enzymatic properties and
industrial applications. In the past 15 years, there has been considerable
progress in the studies of the structure and mechanism of the enzyme but the
focus has shifted to the investigations of the pharmacological actions of the enzyme
and its potential biotechnological and medical applications. Snake venom LAAOs
are interesting multifunctional enzymes exhibiting edema-inducing, platelet
aggregation inhibiting or inducing, apoptotic inducing and anti-HIV-1
activities as well as anticoagulation effect. Their toxicological actions are due
mainly, but not entirely, to the H2O2 liberated during the oxidation. The exact
mechanism of the toxicological actions of snake venom LAAO awaits further
studies. Sun et al. suggested that LAAO may be applied clinically in
glioma therapy by cloning the cDNA of the enzyme and transfect to the tumor
cells of patients to induce the apoptosis in the target tumor cells [49]. Many
authors have demonstrated the apoptotic effect of snake venom LAAO on various
malignant cells (eg, S180 tumor, human breast, acute T cell leukemia, Erlich
ascetic tumor cell lines). There is therefore great potential in the
application of LAAO in cancer therapy. The understanding of the LAAO mode of
action upon parasites may also trigger the design of new drugs or therapeutical
approaches for leishmaniasis as well as other intracellular parasites. In addition,
investigation on the anti-HIV activity of LAAO would also provide valuable
information on the therapeutic development of new generations of anti-HIV
drugs.
Acknowledgement
This work was supported by a research grant Science Fund
02-01-03-SF0153 from the Ministry of Science, Technology and Innovation
(MOSTI), Malaysia.
Snake venom L-amino acid oxidases
Snake venom L-amino acid oxidases 9
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