LAAO

L-amino acid oxidase (LAAO, EC 1.4.3.2) is a flavin protease that specifically oxidizes L-amino acids for deamination to form the α-keto acid, ammonia and H₂O₂ with oxygen consumption. It can be applied to the quantitative analysis of L-amino acids, the resolution of DL-amino acids and the preparation of α-keto acids. In recent years, it has been reported in the literature that LAAO itself has antibacterial, insecticidal, antiviral and other biological activities, and also has the function of interaction with platelets, cytotoxicity and induction of apoptosis. It has extremely broad application prospects in the field of biomedicine.

Figure 1. Representation of the reaction catalyzed by L-amino acid oxidases. (Costa T R, et al. 2014)

Classification

LAAO can be divided into two categories depending on the range of catalytic substrates. One class of LAAO catalyzes a wide range of substrates, such as Rhodococcus erythropolis LAAO, which not only catalyzes 20 L-amino acids, but also catalyzes some of their derivatives. Another class of LAAO catalyzes a narrower range of substrates, such as Rheinheimera LAAO, which acts only strictly on L-lysine. According to the different cofactors, LAAO can also be divided into two categories: snake venom, bacteria and fungi LAAO are dimers and contain 2 FAD cofactors; while rat kidney LAAO have a tetrameric structure and contain 0.5 or 1 FMN cofactor.

Properties

• Substrate specificity and optimality

The substrate specificity and optimality of LAAO from different species are quite different. Some LAAOs can catalyze several or even dozens of amino acids, but some can only catalyze one amino acid. Most LAAOs can only act on L-amino acids, while others can also act on D-amino acids and even amino acid derivatives. Some species contain both widely substrate-specific LAAOs and narrowly substrate-specific LAAOs. Many LAAOs have higher catalytic activity for hydrophobic and neutral amino acids, while some LAAOs have higher activity for other types of amino acids. The study also found that some LAAOs may have different optimal substrates under different conditions.

• Molecular mass and isoelectric point

LAAO has a wide range of sources. The currently known LAAO is generally composed of two non-covalently bonded subunits. Under natural conditions, gel filtration can obtain different molecular masses of LAAO, the range of which is usually 93-150 kDa. The molecular mass of each subunit is generally 36-70 kDa. Some LAAOs consist of two homologous subunits with the same molecular mass, while some LAAOs consist of two non-homologous subunits. The isoelectric point of this enzyme varies widely, ranging from 4.0-8.5 (acidic, medium and alkaline).

• Thermal stability

LAAO has good thermal stability when stored at 4°C and neutral pH. Cobra LAAO has a rapid loss of activity at 70°C, but the enzyme has a certain thermal stability below 60°C, and its thermal denaturation temperature is 65-70°C. LAAO isolated from the eastern spotted rattlesnake has reversible activity, that is, it is inactivated under -20°C, high temperature or strong alkali conditions, and can be restored at pH=5, 37°C. The thermal stability study of Trichoderma viride LAAO showed that it could reach 100% of its original activity at 55°C, 92% of the original activity at 60°C, 42% of the original activity at 65°C, and only 4% of the original activity at 70°C.

Structure

Currently, there have been few reports on the structure of LAAO. Malaysian python LAAO is a dimer. Each monomer has a molecular mass of 55 kDa. It is folded by 15 α-helices and 22 β-sheets to form three domains: FAD binding domain, substrate binding domain and helix domain. A funnel-shaped substrate channel is formed between the substrate binding domain and the helical domain and is combined with the active site. The enzyme has two glycosylation sites, one at Asn172 and the other at Asn361. The prokaryotic Rhodococcus erythropolis LAAO is also a dimer. Each LAAO monomer is composed of 12α-helices, 26β-sheets and 5 3₁₀ helix folds to form three domains: FAD binding domain, substrate binding domain and helix domain.

Figure 2. Structure of l-amino acid oxidase. (A) The functional dimer of LAAO. (B) A single protomer of LAAO.
(Pawelek P D, et al. 2000)

In general, LAAO is first synthesized in the cell as a precursor protein with a signal peptide, and then becomes a mature protein by proteolysis. This may be due to its toxicity, that is, ammonia and H₂O₂ produced by LAAO oxidizing amino acids are toxic to the cells and have a negative impact on cell growth. Therefore, LAAO is first present as a less active precursor in the cell, and then cleaved by endopeptidase into a highly active mature protein that is secreted outside the cell.

Enzyme Activity Assay

At present, there are two commonly used methods for detecting the enzyme activity of LAAO, namely, fluorescence method and spectrophotometry. Both of these methods are based on determining the change amount of H₂O₂ produced by the LAAO catalyzed substrate. Fluorescence method is based on the H₂O₂ produced by enzymatic reaction to convert the non-fluorescent high vanillic acid into a substance with strong fluorescence, and the LAAO enzyme activity is determined according to the change of the fluorescence value of the reaction system. Although the method has high accuracy and sensitivity, the fluorescence lifetime is short and it is easy to be quenched. It is necessary to avoid prolonged exposure to light and air during operation. The spectrophotometry is based on the H₂O₂ produced by enzymatic reaction which acts on the pigmentary substance to produce an absorbance at a certain wavelength, and the LAAO enzyme activity is measured according to the change in the absorbance of the reaction system. This method has the obvious advantages of fast response, simple operation, good repeatability, and low price. Therefore, spectrophotometry is the most commonly used method for determining enzyme activity of LAAO.

Biological Functions

• Inducing apoptosis

Studies have shown that snake venom LAAO can induce apoptosis of a variety of cells, among which, there are many reports of induction of apoptosis of vascular endothelial cells. Studies have shown that LAAO can inhibit the growth of human umbilical vein endothelial cells and affect the formation of cell tubules in a dose-dependent manner. A large number of experiments have confirmed that proliferation, migration and tubule formation of vascular endothelial cells are key steps in tumor growth. Inhibition of angiogenesis is expected to be a breakthrough point in the treatment of cancer, so the researchers speculate that LAAO has the effect of inhibiting tumor angiogenesis. In the induction of apoptosis, H₂O₂ produced by snake venom LAAO catalysis plays a major role. H₂O₂ up-regulates the expression of the pro-apoptotic gene Fas and causes apoptosis. Therefore, LAAO-induced apoptosis may be due to the H₂O₂ produced by LAAO catalyzing L-amino acids.

• Bacteriostatic effect

It has long been known that rattlesnake poison has the effect of resisting Gram-positive bacteria, and studies have shown that LAAO plays an antibacterial role. LAAO can adhere to the cell surface of Escherichia coli and Bacillus subtilis and produce high concentrations of H₂O₂, and this bacteriostatic effect is dose-dependent. The more LAAO added to the medium, the stronger the inhibition of bacterial growth. Experiments have shown that a small amount of peroxidase can inhibit the bacteriostasis of LAAO. The sensitivity of different bacteria to LAAO may be due to the fact that different bacteria produce unequal amounts of peroxidase, resulting in different degrees of offset to oxidation.

• Effect on platelets

Studies have shown that the role of snake venom LAAO on platelets has two conflicting results: inhibition or induction of platelet aggregation. A large amounts of literature indicates that LAAO inhibits platelet aggregation induced by agonists and shear forces. For example, LAAO isolated and purified from the snake venom of Echis colorata can dose-suppress platelet aggregation caused by agonist (adenosine diphosphate, ADP). However, there are also reports that the cobra venom LAAO can induce platelet aggregation in platelet-rich plasma, and that sarcosine phosphate and sarcosine phosphokinase cannot inhibit platelet aggregation induced by the colobus LAAO. We cannot explain this contradictory conclusion accurately. But one thing is certain, H₂O₂ plays an important role in these two opposite roles.

References

1. Pawelek P D, et al. The structure of L-amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site. [J]. Embo Journal, 2000, 19(16):4204–4215.
2. Costa T R, et al. Snake venom L-amino acid oxidases: an overview on their antitumor effects. [J]. Journal of Venomous Animals and Toxins including Tropical Diseases, 2014, 20(1):23.