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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:




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).





L-Amino acid oxidase (L-amino acid:O2 oxidoreductase, E.C. 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




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.




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.





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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