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Mucus layer that covers the body surface of various animal functions as a defense barrier against microbes, environmental xenobiotics, and predators. Previous studies have reported that L-amino acid oxidase (LAAO), present in several animal fluids, has potent properties against pathogenic bacteria, viruses, and parasites. LAAO catalyzes the oxidative deamination of specific L-amino acids with the generation of hydrogen peroxide and L-amino acid metabolites. Further, the generated hydrogen peroxide is involved in oxidation (direct effect) while the metabolites activate immune responses (indirect effect). Therefore, LAAO exhibits two different mechanisms of bioactivation. Previously, we described the selective, specific, and local oxidative and potent antibacterial actions of various LAAOs as potential therapeutic strategies. In this review, we focus on their biochemical features, enzymatic regulations, and biomedical applications with a view of describing their probable role as biochemical agents and biomarkers for microbial infections, cancer, and autoimmune-mediated diseases. We consider that LAAOs hold implications in biomedicine owing to their antimicrobial activity wherein they can be used in treatment of infectious diseases and as diagnostic biomarkers in the above-mentioned diseased conditions. •Focus on biochemical features, enzymatic regulation, and biomedical applications of LAAOs. •Mechanisms of antimicrobial activity, inflammatory regulation, and immune responses of LAAOs. •Potential biomedical application as an antimicrobial and anti-infection agent, and disease biomarker.

The venom color variation of Crotalus durissus terrificus (Cdt) is attributed to the presence of the toxin L-amino acid oxidase (LAAO). During the venom milking routine of Instituto Butantan, we have noticed that most venoms of captive Cdt specimens show a yellowish color, while most venoms of wild specimens are white. Here we describe a comparative analysis of long-term captive (LTC) and recently wild-caught (RWC) Cdt, focusing on LAAO variation. For the identification of LAAO in individual venoms, four different approaches were employed: evaluation of the enzymatic activity, SDS-PAGE, Western blotting, and ELISA. In addition, mass spectrometry analysis was performed using pooled samples. Although some variation among these methodologies was observed, it was possible to notice that the presence of LAAO was significantly higher in the venom of LTC individuals. LAAO was identified in 60–80% LTC specimens and in only 10–12% of RWC specimens. Furthermore, this enzyme accounts for 5.6% of total venom proteins of LTC Cdt pooled venom, while it corresponds to only 0.7% of RWC Cdt pooled venom. These findings strongly suggest that captive maintenance increases the expression of LAAO in Cdt venom.

An l -amino acid oxidase (LAO) is an amino acid metabolism enzyme that also performs a variety of biological activities. Recently, LAOs have been discovered to be deeply involved in innate immunity in fish because of their antibacterial and antiparasitic activity. The determinant of potent antibacterial/antiparasitic activity is the H2O2 byproduct of LAO enzymatic activity that utilizes the l -amino acid as a substrate. In addition, fish LAOs are upregulated by pathogenic bacteria or parasite infection. Furthermore, some fish LAOs show that the target specificity depends on the virulence of the bacteria. All results reflect that LAOs are new innate immune molecules. This review also describes the potential of the immunomodulatory functions of fish LAOs, not only the innate immune function by a direct oxidation attack of H2O2.

L-amino acid oxidases (LAAOs) have antibacterial activity and play important roles in innate immunity. We have previously identified a LAAO of ~52 kDa in size from the mucus layer of the flounder Platichthys stellate (psLAAO1) and have successfully produced psLAAO1 as a secreted bioactive recombinant protein by using Pichia pastoris (P. pastoris). The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in the mucus layer. In this study, homology modeling of psLAAO1 predicted metal coordination by residues Y241, H348, and D406. We show that the Michaelis constant (Km) of psLAAO1 decreased and the catalytic constant (Kcat/Km) value increased following pre-treatment of the protein with a chelating agent. In contrast to the non-chelated protein sample, enzymatic activity of EDTA-treated psLAAO1 gradually decreased or was absent after one or two freeze-thaw cycles. The H348A psLAAO1 mutant generated by site-directed mutagenesis and recombinantly produced by P. pastoris did not display antibacterial activity. The results of the metal detection assay revealed that for the non-metal coordinating histidine mutant (H209A, control), the levels of iron, zinc, and magnesium were similar to those of wild-type psLAAO1, whereas magnesium was not detected in the H348A mutant sample. A wild-type psLAAO1 sample treated with chelating agent did not contain zinc and magnesium ions. In conclusion, metal coordination by psLAAO1 affects enzymatic activity, and H348 is involved in the coordination of magnesium, and metal coordination by psLAAO1 provides essential structural stability. KEY POINTS: • Homology modeling of psLAAO1 predicted metal coordination by residue H348 • The H348A psLAAO1 mutant showed no antibacterial activity or magnesium coordination • Metal coordination by H348 affects enzyme activity and structural stability.

Bothrops brazili is a pitviper from Amazonian region, responsible for many accidents in Peru. Despite its relevance, its venom has not been extensively characterized. In the present work, Bothrops brazili venom (BbV) components were analyzed by RP-HPLC, SDS-PAGE and MALDI-TOF/TOF. Approximately 37 proteins were identified, belonging to 7 families. Snake venom metalloproteinases (SVMPs) were the most abundant proteins of the venom (33.05%), followed by snake venom serine proteinases (SVSPs, 26.11%), phospholipases A2 (PLA2, 25.57%), snake C-type lectins (CTLs, 9.61%), L-aminoacid oxidase (LAAO, 3.80%), cystein-rich secretory proteins (CRISP, 1.67%) and Bradykinin-potentiating peptide (BPP, 0.20%). In vitro enzymatic activities of BbV showed high levels of SVMP activity and reduced Hyal activity in comparison with other bothropic venoms. Furthermore, BbV reduced VERO cells viability. ELISA and Western Blotting showed that both Peruvian and Brazilian bothropic antivenoms were able to recognize BbV components. This work provides an overview of BbV venom content and indicates a potential efficiency of Peruvian and Brazilian antivenoms to treat accidents with this species.

l-tryptophan (Trp) is widely used in food and feed enforcement to play an important role in biological processes. Various metabolites of Trp perform its potent function. The indole pyruvate pathway is one of the main pathways of Trp metabolism in the gut microbiota, providing numerous indole-derivatives, which can modulate intestinal homeostasis and mucosal immunity by activating the aryl hydrocarbon receptor (AHR) signaling pathway. In this study, we constructed an IL4I1-overexpressed 293T cell line and found that IL4I1 can catalyze Trp to produce indole-3-acetic acid (IAA) and indole-3-carboxaldehyde (IAld). Moreover, both IAA and IAld are accumulated in dendritic cells (DCs) and can stimulate the expression of CYP1A1. Our results demonstrate the existence of the indole pyruvate pathway in host cells with IL4I1 as the key enzyme. The IL4I1-mediated Trp metabolism implies the role of dietary impact on immunity.

l-Amino acid oxidase (LAAO) is widely distributed in nature and shows important biological activity. It induces cell apoptosis and has antibacterial properties. This study was designed to investigate the effect of methyl substituent on its activity as methylated derivatives of l-tyrosine, labelled with short-lived B+ emitters, have been used in oncological diagnostics. To study isotope effects in the oxidative deamination of O-methyl-l-tyrosine, the deuterated isotopomer, i.e. O-methyl-[2-2H]-l-tyrosine, was synthesized by isotope exchange, catalyzed enzymatically by tryptophanase. Isotope effects were determined using the spectrophotometric non-competitive method. The values of isotope effects indicate that the α-C–H bond cleavage occurs in the rate determining step of the investigated reaction and α-hydrogen plays a role in the substrate binding process at the enzyme active site. The inhibitory effect on LAAO activity was studied with α-methyl-l-tyrosine and N-methyl-l-tyrosine. The mode of inhibition was determined based on Lineweavear–Burk plots intersections. α-Methyl-l-tyrosine has been found a mixed type inhibitor of the investigated enzyme, whereas N-methyl-l-tyrosine is a non-competitive inhibitor of LAAO.

The phospholipase A2 (PLA2) and l-amino acid oxidase (LAAO) are two major enzymes found in the venoms from most snake species. These enzymes have been structurally and functionally characterised for their pharmacological activities. Both PLA2 and LAAO from different venoms demonstrate considerable cytotoxic effects on cancer cells via induction of apoptosis, cell cycle arrest and suppression of proliferation. These enzymes produce more pronounced cytotoxic effects in cancer cells than normal cells, thus they can be potential sources as chemotherapeutic agents. It is proposed that PLA2 and LAAO contribute to an elevated oxidative stress due to their catalytic actions, for instance, the ability of PLA2 to produce reactive oxygen species during lipolysis and formation of H2O2 from LAAO catalytic activity which consequently lead to cell death. Nonetheless, the cell-death signalling pathways associated with exposure to these enzymatic toxins are not fully elucidated yet. Here in this review, we will discuss the cytotoxic effects of PLA2 and LAAO in relationship to their catalytic mechanisms and the underlying mechanisms of cytotoxic actions.

The venomic profile of Asian mountain pit viper Ovophis monticola is clarified in the present study. Using mass spectrometry-based proteomics, 247 different proteins were identified in crude venom of O. monticola found in Thailand. The most abundant proteins were snake venom metalloproteases (SVMP) (36.8%), snake venom serine proteases (SVSP) (31.1%), and phospholipases A2 (PLA2) (12.1%). Less abundant proteins included L-amino acid oxidase (LAAO) (5.7%), venom nerve growth factor (3.6%), nucleic acid degrading enzymes (3.2%), C-type lectins (CTL) (1.6%), cysteine-rich secretory proteins (CRISP) (1.2%) and disintegrin (1.2%). The immunoreactivity of this viper’s venom to a monovalent antivenom against green pit viper Trimeresurus albolabris, or to a polyvalent antivenom against hemotoxic venom was investigated by indirect ELISA and two-dimensional (2D) immunoblotting. Polyvalent antivenom showed substantially greater reactivity levels than monovalent antivenom. A titer for the monovalent antivenom was over 1:1.28x107 dilution while that of polyvalent antivenom was 1:5.12x107. Of a total of 89 spots comprising 173 proteins, 40 spots of predominantly SVMP, SVSP and PLA2 were specific antigens for antivenoms. The 49 unrecognized spots containing 72 proteins were characterized as non-reactive proteins, and included certain types of CTLs and CRISPs. These neglected venom constituents could limit the effectiveness of antivenom-based therapy currently available for victims of pit viper envenomation.

l -amino acid oxidases (LAOs) catalyze the oxidative deamination of l -amino acid and generate α-keto acid, ammonia, and hydrogen peroxide as byproducts. LAOs showed the variety of bioactivity by the resulting hydrogen peroxide. The serum of the red-spotted Epinephelus akaara contains an LAO (Ea-LAO) with the potential to kill bacterial pathogens Aeromonas salmonicida and Vibrio anguillarum via hydrogen peroxide. However, it is unknown how the grouper tolerates the harmful effects of the serum Ea-LAO byproducts. In this study, we analyzed the kinetics of fish LAOs to understand how they escape the toxicity of byproducts. The LAO activity of grouper serum was suppressed in low-salt solutions such as NaCl, CaCl2, MgCl2, and diluted seawater. The activity was non-linearly increased and fitted to the four-parameter log-logistic model. The EC50 of the seawater was calculated to have a 0.72-fold concentration. This result suggested that the Ea-LAO could be activated by mixing with seawater. Circular dichroism spectrometry showed that the α helix content was estimated to be 12.1% and 5.3% in a salt-free buffer (inactive condition) and the original concentration of seawater (active condition), respectively, indicating that the secondary structure of the Ea-LAO in the active condition was randomized. In addition, the Ea-LAO showed reversible LAO activity regulation according to the salt concentration in the environment. Taken together, this indicates that the Ea-LAO is normally on standby as an inactive form, and it could activate as a host-defense molecule to avoid pathogen invasion via a wound when mixed with seawater.

The effect of Micrurus mipartitus snake venom as a therapeutic alternative for T-acute lymphoblastic leukemia (ALL) is still unknown. This study was aimed to evaluate the cytotoxic effect of M. mipartitus snake venom and a new L-amino acid oxidase (LAAO), named MipLAAO, on human peripheral blood lymphocytes (PBL) and on T-ALL cells (Jurkat), and its mechanism of action. PBL and Jurkat cells were treated with venom and MipLAAO, and morphological changes in the cell nucleus/DNA, mitochondrial membrane potential, levels of intracellular reactive oxygen species and cellular apoptosis markers were determined by fluorescence microscopy, flow cytometry and pharmacological inhibition. Venom and MipLAAO induced apoptotic cell death in Jurkat cells, but not in PBL, in a dose-response manner. Additionally, venom and MipLAAO increased dichlorofluorescein fluorescence intensity, indicative of H2O2 production, increased DJ-1 Cys106-sulfonate, as a marker of intracellular stress and induced the up-regulation of PUMA, p53 and phosphorylation of c-JUN. Additionally, it increased the expression of apoptotic CASPASE-3. In conclusion, M. mipartitus venom and MipLAAO selectively induces apoptosis in Jurkat cells through a H2O2-mediated signaling pathway dependent mostly on CASPASE-3 pathway. Our findings support the potential use of M. mipartitus snake venom compounds as a potential treatment for T-ALL.

l-Amino acid oxidases (LAAOs) are flavoproteins, which use oxygen to deaminate l-amino acids and produce the corresponding α-keto acids, ammonia, and hydrogen peroxide. Here we describe the heterologous expression of LAAO4 from the fungus Hebeloma cylindrosporum without signal sequence as fusion protein with a 6His tag in Escherichia coli and its purification. 6His-hcLAAO4 could be activated by exposure to acidic pH, the detergent sodium dodecyl sulfate, or freezing. The enzyme converted 14 proteinogenic l-amino acids with l-glutamine, l-leucine, l-methionine, l-phenylalanine, l-tyrosine, and l-lysine being the best substrates. Methyl esters of these l-amino acids were also accepted. Even ethyl esters were converted but with lower activity. Km values were below 1 mM and vmax values between 19 and 39 U mg−1 for the best substrates with the acid-activated enzyme. The information for an N-terminal aldehyde tag was added to the coding sequence. Co-expressed formylglycine-generating enzyme was used to convert a cysteine residue in the aldehyde tag to a Cα-formylglycine residue. The aldehyde tag did not change the properties of the enzyme. Purified Ald-6His-hcLAAO4 was covalently bound to a hexylamine resin via the Cα-formylglycine residue. The immobilized enzyme could be reused repeatedly to generate phenylpyruvate from l-phenylalanine with a total turnover number of 17,600 and was stable for over 40 days at 25 °C.

Neutrophils play a pivotal role in innate immunity and in the inflammatory response. Neutrophils are very motile cells that are rapidly recruited to the inflammatory site as the body first line of defense. Their bactericidal activity is due to the release into the phagocytic vacuole, called phagosome, of several toxic molecules directed against microbes. Neutrophil stimulation induces release of this arsenal into the phagosome and induces the assembly at the membrane of subunits of the NAPDH oxidase, the enzyme responsible for the production of superoxide anion that gives rise to other reactive oxygen species (ROS), a process called respiratory burst. Altogether, they are responsible for the bactericidal activity of the neutrophils. Excessive activation of neutrophils can lead to extensive release of these toxic agents, inducing tissue injury and the inflammatory reaction. Envenomation, caused by different animal species (bees, wasps, scorpions, snakes etc.), is well known to induce a local and acute inflammatory reaction, characterized by recruitment and activation of leukocytes and the release of several inflammatory mediators, including prostaglandins and cytokines. Venoms contain several molecules such as enzymes (phospholipase A2, L-amino acid oxidase and proteases, among others) and peptides (disintegrins, mastoporan, parabutoporin etc.). These molecules are able to stimulate or inhibit ROS production by neutrophils. The present review article gives a general overview of the main neutrophil functions focusing on ROS production and summarizes how venoms and venom molecules can affect this function.

International audience; The amino-acid catabolizing enzyme Interleukin-4 induced gene 1 (IL4I1) remains poorly characterized despite it is emerging as a pertinent therapeutic target for cancer. IL4I1 is secreted in the synaptic cleft by antigen-presenting cells. It inhibits TCR signaling, modulates naïve T cell differentiation and limits effector T cell proliferation. IL4I1 expression in tumors shapes the tumor microenvironment and impairs the antitumor cytotoxic T cell response, thereby facilitating cancer immune escape. Several mechanisms participate in these effects. Recent data suggest a role of new IL4I1 metabolites in activation of the aryl-hydrocarbon receptor (AHR). Here, we observe that expression of IL4I1 is poorly correlated with that of validated targets of AHR in human cancers. Moreover, dendritic cells do not upregulate AHR target genes in relation with IL4I1 expression in vivo. Finally, IL4I1 activity towards tryptophan leading to production of AHR-activating products is very low, and should be negligible when tryptophan-degrading enzymes of higher affinity compete for the substrate. We recently showed that IL4I1 expression by dendritic cells directly regulates immune synapse formation and modulates the repertoire and memory differentiation of responding CD8 T cells after viral infection. Thus, IL4I1 may restrain tumor control through regulating the priming of tumor-specific CD8 T cells, independently of AHR activation.

This study evaluated the potential of antitumor activity of snake venom from Vipera ammodytes and L-amino acid oxidase from Crotalus adamanteus on different colorectal cancer cell lines through determination of cytotoxic activity by MTT assay, pro-apoptotic activity by acridine orange/ethidium bromide staining, and concentrations of redox status parameters (superoxide, reduced glutathione, lipid peroxidation) by colorimetric methods. The expression of genes involved in the biotransformation process and metabolite efflux was determined by qPCR method, while protein expression of glutathione synthetase and P-glycoprotein were analysed by immunocytochemistry. The analysis of cell death shows that snake venom dominantly leads cells to necrosis. Induction of apoptosis by L-amino acid oxidase was in correlation with oxidative disbalance in cancer cells. Gene expression profile of membrane transporters and CYP genes were different in each cell line and in correlation with their sensitivity of treatment. Our results show that L-amino acid oxidase from snake venom is a potent cytotoxic substance with pronounced pro-apoptotic activity. The inhibition of P-glycoprotein suggests that L-amino acid oxidase is a good substance for furter research of antitumor effect, with unexpressed potential for occurrence of drug resistance in vitro.

A large number of protein sequences are registered in public databases such as PubMed. Functionally uncharacterized enzymes are included in these databases, some of which likely have potential for industrial applications. However, assignment of the enzymes remained difficult tasks for now. In this study, we assigned a total of 28 original sequences to uncharacterized enzymes in the FAD-dependent oxidase family expressed in some species of bacteria including Chryseobacterium, Flavobacterium, and Pedobactor. Progenitor sequence of the assigned 28 sequences was generated by ancestral sequence reconstruction, and the generated sequence exhibited L-lysine oxidase activity; thus, we named the enzyme AncLLysO. Crystal structures of ligand-free and ligand-bound forms of AncLLysO were determined, indicating that the enzyme recognizes L-Lys by hydrogen bond formation with R76 and E383. The binding of L-Lys to AncLLysO induced dynamic structural change at a plug loop formed by residues 251 to 254. Biochemical assays of AncLLysO variants revealed the functional importance of these substrate recognition residues and the plug loop. R76A and E383D variants were also observed to lose their activity, and the kcat/Km value of G251P and Y253A mutations were approximately 800- to 1800-fold lower than that of AncLLysO, despite the indirect interaction of the substrates with the mutated residues. Taken together, our data demonstrate that combinational approaches to sequence classification from database and ancestral sequence reconstruction may be effective not only to find new enzymes using databases of unknown sequences but also to elucidate their functions.

Interleukin-4-induced-1 (IL4i1) is an amino acid oxidase secreted from immune cells. Recent observations have suggested that IL4i1 is pro-tumorigenic via unknown mechanisms. As IL4i1 has homologs in snake venoms (L-amino acid oxidases [LAAO]), we used comparative approaches to gain insight into the mechanistic basis of how conserved amino acid oxidases regulate cell fate and function. Using mammalian expressed recombinant proteins, we found that venom LAAO kills cells via hydrogen peroxide generation. By contrast, mammalian IL4i1 is non-cytotoxic and instead elicits a cell protective gene expression program inhibiting ferroptotic redox death by generating indole-3-pyruvate (I3P) from tryptophan. I3P suppresses ferroptosis by direct free radical scavenging and through the activation of an anti-oxidative gene expression program. Thus, the pro-tumor effects of IL4i1 are likely mediated by local anti-ferroptotic pathways via aromatic amino acid metabolism, arguing that an IL4i1 inhibitor may modulate tumor cell death pathways.

Recently, l-amino acid oxidases (LAAOs) have been identified in several fish species as first-line defense molecules against bacterial infection. Here, we report the cloning and characterization of a fish LAAO gene, EcLAAO2, from orange-spotted grouper (Epinephelus coioides). The full-length cDNA is 3030 bp, with an ORF encoding a protein of 511 amino acids. EcLAAO2 is mainly expressed in the fin, gill, and intestine. Its expression is upregulated in several immune organs after challenge with lipopolysaccharide (LPS) and poly (I:C). The recombinant EcLAAO2 protein (rEcLAAO2), expressed and purified from a baculovirus expression system, was determined to be a glycosylated dimer. According to a hydrogen peroxide-production assay, the recombinant protein was identified as having LAAO enzyme activity with substrate preference for L-Phe and L-Trp, but not L-Lys as other known fish LAAOs. rEcLAAO2 could effectively inhibit the growth of Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis while exhibiting less effective inhibition of the growth of Escherichia coli. Finally, protein models based on sequence homology were constructed to predict the three-dimensional structure of EcLAAO2 as well as to explain the difference in substrate specificity between EcLAAO2 and other reported fish LAAOs. In conclusion, this study identifies EcLAAO2 as a novel fish LAAO with a substrate preference distinct from other known fish LAAOs and reveals that it may function against invading pathogens.

IL4I1 is an immunoregulatory enzyme that inhibits CD8 T-cell proliferation in vitro and in the tumoral context. Here, we dissected the effect of IL4I1 on CD8 T-cell priming by studying the differentiation of a transgenic CD8 T-cell clone and the endogenous repertoire in a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. Unexpectedly, we show that IL4I1 accelerates the expansion of functional effector CD8 T cells during the first several days after infection and increases the average affinity of the elicited repertoire, supporting more efficient LCMV clearance in WT mice than IL4I1-deficient mice. Conversely, IL4I1 restrains the differentiation of CD8 T-cells into long-lived memory precursors and favors the memory response to the most immunodominant peptides. IL4I1 expression does not affect the phenotype or antigen-presenting functions of dendritic cells (DCs), but directly reduces the stability of T-DC immune synapses in vitro, thus dampening T-cell activation. Overall, our results support a model in which IL4I1 increases the threshold of T-cell activation, indirectly promoting the priming of high-affinity clones while limiting memory T-cell differentiation.

The fungal glycoprotein l-lysine &alpha, oxidase (LO) catalyzes the oxidative deamination of l-lysine (l-lys). LO may be internalized in the intestine and shows antitumor, antibacterial, and antiviral effects in vivo. The main mechanisms of its effects have been shown to be depletion of the essential amino acid l-lys and action of reactive oxidative species produced by the reaction. Here, we report that LO penetrates into the brain and is retained there for up to 48 h after intravenous injection, which might be explained by specific pharmacokinetics. LO actively intervenes in amino acid metabolism in the brain. The most significant impact of LO was towards amino acids, which are directly exposed to its action (l-lys, l-orn, l-arg). In addition, the enzyme significantly affected the redistribution of amino acids directly associated with the tricarboxylic acid (TCA) cycle (l-asp and l-glu). We discovered that the depletion of l-orn, the precursor of polyamines (PA), led to a significant and long-term decrease in the concentration of polyamines, which are responsible for regulation of many processes including cell proliferation. Thus, LO may be used to reduce levels of l-lys and PA in the brain.

Ammonia is critical for agricultural and chemical industries. The extracellular production of ammonia by yeast (Saccharomyces cerevisiae) using cell surface engineering can be efficient approach because yeast can avoid growth deficiencies caused by knockout of genes for ammonia assimilation. In this study, we produced ammonia outside the yeast cells by displaying an l-amino acid oxidase with a wide substrate specificity derived from Hebeloma cylindrosporum (HcLAAO) on yeast cell surfaces. The HcLAAO-displaying yeast successfully produced 12.6 m m ammonia from a mixture of 20 proteinogenic amino acids (the theoretical conversion efficiency was 63%). We also succeeded in producing ammonia from a food processing waste, soybean residues (okara) derived from tofu production. The conversion efficiency was 88.1%, a higher yield than reported in previous studies. Our study demonstrates that ammonia production outside of yeast cells is a promising strategy to utilize food processing wastes.

This study aims to examine whether two L-amino acid oxidases isolated from Bothrops snake venom (SV-LAAOs) were cytotoxic to Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis, two causative agents of leishmaniasis, which is an endemic disease in tropical and subtropical countries. The SV-LAAOs BjussuLAAO-II and BmooLAAO-II were isolated from Bothrops jararacussu and Bothrops moojeni venom, respectively, through a three-step chromatography process that used molecular exclusion, hydrophobic interaction, and affinity columns. BmooLAAO-II is a new SV-LAAO isoform that we isolated in this study. The purified BjussuLAAO-II and BmooLAAO-II had high L-amino acid oxidase-specific activity: 3481.17 and 4924.77 U/mg/min, respectively. Both SV-LAAOs were strongly cytotoxic to the two Leishmania species, even at low concentrations. At the same concentration, BjussuLAAO-II and BmooLAAO-II exerted different cytotoxic effects on the parasites. We reported for the first time that the SV-LAAOs suppressed cell proliferation and altered the mitochondrial membrane potential of the two Leishmania species. Surprisingly, BjussuLAAO-II increased the intracellular reactive oxygen species production only in L. (L.) amazonensis, while BmooLAAO-II increased the intracellular reactive oxygen species production only in L. (V.) braziliensis, indicating that these SV-LAAOs had a certain specificity of action.

Intra-specific venom variation has the potential to provide important insights into the evolution of snake venom, but remains a relatively neglected aspect of snake venom studies. We investigated the venom from 13 individual coastal taipans Oxyuranus scutellatus from four localities on the north-east coast of Australia, spanning a distance of 2000km. The intra-specific variation in taipan venom was considerably less than the inter-specific variation between it and the other Australian elapids to which it was compared. The electrophoretic venom profile of O. scutellatus was visually different to six other genera of Australian elapids, but not to its congener inland taipan O. microlepidotus. There was minimal geographical variation in taipan venom, as the intra-population variation exceeded the inter-population variation for enzymatic activity, procoagulant activity, and the abundance of neurotoxins. The pre-synaptic neurotoxin (taipoxin) was more abundant than the post-synaptic neurotoxins (3FTx), with a median of 11.0% (interquartile range (IQR): 9.7% to 18.3%, range: 6.7% to 23.6%) vs. a median of 3.4% (IQR: 0.4% to 6.7%, range: 0% to 8.1%). Three taipan individuals almost completely lacked post-synaptic neurotoxins, which was not associated with geography and occurred within two populations. We found no evidence of sexual dimorphism in taipan venom. Our study provides a basis for evaluating the significance of intra-specific venom variation within a phylogenetic context by comparing it to the inter-specific and inter-generic variation. The considerable intra-population variation we observed supports the use of several unpooled individuals from each population when making inter-specific comparisons.

In fish, the innate immune system is more important than the adaptive immune system because it responds quickly and nonspecifically to protect against pathogens. Thus, a variety of innate immune molecules have been found in fish. Recently, l-amino acid oxidases (LAOs) were discovered as a new member of the antibacterial protein from fish skin mucus and serum. In this study, we newly found an antibacterial LAO in red-spotted grouper (Epinephelus akaara) serum. It showed a broad range of substrate specificity with aromatic and hydrophobic amino acids. The grouper LAO gene had a low expression level in the kidney under normal conditions; however, it was significantly upregulated by blood loss 1 day after bleeding. In addition, the LAO activity in the serum recovered within 3 days in the same experiment. This quick recovery may indicate that the LAO is an essential innate immune molecule in the whole grouper body.

Objectives Snakebite envenoming is an important public health problem that threatens the lives of healthy individuals especially in many tropical countries like Nigeria. Antivenins, the only efficient approach for snakebite envenoming, are limited in their efficacy in the neutralization of local tissue damage. Snake venom phospholipase A2 (PLA2), protease, hyaluronidase and l-amino acid oxidase (LAAO) are the major hydrolytic enzymes involve in local tissue damage. Therefore, this study evaluates the inhibitory effect of kolaviron (KV) against Naja n. nigricollis (NNN) snake venom hydrolytic enzymes involved in local tissue damage. Methods Kolaviron was evaluated for its ability to inhibit the hydrolytic enzyme activities of NNN venom phospholipase A2 (PLA2), protease, hyaluronidase and l-amino acid oxidase (LAAO). Present study also deals with the neutralization of NNN venom enzyme(s) induced complications such as myotoxic, edemic, hemolytic and procoagulant effects. Results Kolaviron inhibited the PLA2, protease, hyaluronidase and LAAO enzyme activities of NNN venom in a dose-dependent manner. Furthermore, myotoxic, edemic, hemolytic and procoagulant effects induced by NNN venom enzyme were neutralized significantly (p Conclusions These findings clearly present kolaviron as a potent inhibitor against NNN venom hydrolytic enzymes involved in local tissue damage and may act by either forming an inhibitor-enzyme complex that restricts the substrate availability to the enzyme or direct binding to the enzyme active site that affects the enzyme activity thereby mitigating venom-induced toxicity.

Ralstonia solanaceraum is the quarantine plant pathogenic bacterium that causes bacterial wilt in over 200 host plants, which include economically important crops such as potato, tomato, tobacco, banana, and ginger. Alternative biological methods of disease control that can be used in integrated pest management are extensively studied. In search of new proteins with antibacterial activity against R. solanacearum, we identified L-amino acid oxidases (LAOs) from fruiting bodies of Amanita phalloides (ApLAO) and Infundibulicybe geotropa (CgLAO). We describe an optimized isolation procedure for their biochemical characterization, and show that they are dimeric proteins with estimated monomer molecular masses of 72 and 66 kDa, respectively, with isoelectric point of pH 6.5. They have broad substrate specificities for hydrophobic and charged amino acids, with highest Km for L-Leu, and broad pH optima at pH 5 and pH 6, respectively. An enzyme with similar properties is also characterized from the mycelia of I. geotropa (CgmycLAO). Fractionated aqueous extracts of 15 species of mushrooms show that LAO activity against L-Leu correlates with antibacterial activity. We confirm that the LAO activities mediate the antibacterial actions of ApLAO, CgLAO, and CgmycLAO. Their antibacterial activities are greater against Gram-negative versus Gram-positive bacteria, with inhibition of growth rate, prolongation of lag-phase, and decreased endpoint biomass. In Gram-positive bacteria, they mainly prolong the lag phase. These in vitro antibacterial activities of CgLAO and CgmycLAO are confirmed in vivo in tomato plants, while ApLAO has no effect on disease progression in planta. Transmission electron microscopy shows morphological changes of R. solanacearum upon LAO treatments. Finally, broad specificity of the antibacterial activities of these purified LAOs were seen for in vitro screening against 14 phytopathogenic bacteria. Therefore, these fungal LAOs show great potential as new biological phytoprotective agents and show the fruiting bodies of higher fungi to be a valuable source of antimicrobials with unique features.

Bacillus subtilis HLZ-68 can produce l-amino acid oxidase (l-AAO), and dl-arginine can be degraded asymmetrically by suspending the wet bacterial biomass in the degradation liquid. By adding oxygen-vectors to the fermentation medium, the collected amount of wet bacterial biomass can be increased. Taking n-dodecane, n-hexadecane, oleic acid, paraffin and n-hexane as oxygen-vectors, the optimal oxygen-vector was 1.2% (v/v) oleic acid. The wet weight biomass increased by 66.83% and the activity of l-AAO in the fermentation broth increased by 38.88% compared with those before the addition of oxygen-vector. The standard sample dl-arginine was derivatized by phenyl isothiocyanate, and then subjected to high-performance liquid chromatography (HPLC), and the obtained peak area and arginine content were used as standard curves to measure the dl-arginine. The content of d-arginine and l-arginine in the initial degradation solution was 50% each, and the bacterial cells were added to the initial degradation solution of dl-arginine. After 21 h of reaction, l-arginine was completely degraded, leaving 47% of d-arginine. d-alanine was easily extracted from the reaction solution using cation-exchange resin, after centrifugation, decolourization, concentration and vacuum drying, and the chemical and optical purity of the extracted d-arginine were 92.68% and 97.46%, respectively.

The biocatalytic oxidation of amino acids represents an attractive approach towards the synthesis of alpha-keto acids, which are interest for various industrial applications. As l-amino acids are readily available from fermentation processes, these natural amino acids can serve as substrates in combination with an L-amino acid oxidase. Besides an aqueous phase as reaction medium, a further advantage of such a process is the utilization of air as oxidation agent. In this study, we studied the organic-synthetic properties of a literature-known recombinant L-amino acid oxidase from the fungus Hebeloma cylindrosporum with respect to its suitability to catalyze the formation of alphaketo acids exemplified for the synthesis of phenylpyruvic acid starting from L-phenylalanine as a substrate. In our study the enzyme displayed a reasonable operational stability in the reaction system and as well as promising applicability data with respect to substrate and product inhibition. In a biotransformation, 20mM of substrate were converted after 4hours reaction. The formation of undesired by-products was suppressed using a commercially available catalase enzyme. Copyright © 2020. Published by Elsevier B.V.

l‐amino acid oxidases (LAAOs) are flavoenzymes that catalyze the oxidative deamination of l‐amino acids to the corresponding α‐keto acids, ammonia, and hydrogen peroxide. Here, we show the overexpression, purification, and the characterization of LAAO4 from the fungus Hebeloma cylindrosporum in the yeast Pichia pastoris with a 9His‐tag and compare this with the recently characterized 6His‐hcLAAO4 expressed in E. coli. The expression of the enzyme with an ER‐signal sequence in P. pastoris resulted in a glycosylated, secreted protein. The enzymatic activity without activation was higher after expression in P. pastoris compared to E. coli. Due to treatment with acidic pH, a striking increase of activity could be detected for both expression systems resulting in similar specific activities after acid activation. Regarding the substrate spectrum, temperature stability, K m, and v max values, hcLAAO4 showed very few differences when produced in these two expression systems. A higher yield of hcLAAO4 could be obtained by fermentation., hcLAAO4 was successfully expressed in Pichia pastoris as a secreted, glycosylated enzyme. K m and v max values, substrate spectrum, and temperature stability were similar when expressed in E. coli or in P. pastoris. Expression of hcLAAO4 could be drastically increased when fermented in bioreactors compared to shaking flasks in both expression systems with much higher values obtained regarding activity per gram dry mass and activity per liter for P. pastoris.

Background The cellular response to ionizing radiation involves activation of p53-dependent pathways and activation of the atypical NF-κB pathway. The crosstalk between these two transcriptional networks include (co)regulation of common gene targets. Here we looked for novel genes potentially (co)regulated by p53 and NF-κB using integrative genomics screening in human osteosarcoma U2-OS cells irradiated with a high dose (4 and 10 Gy). Radiation-induced expression in cells with silenced TP53 or RELA (coding the p65 NF-κB subunit) genes was analyzed by RNA-Seq while radiation-enhanced binding of p53 and RelA in putative regulatory regions was analyzed by ChIP-Seq, then selected candidates were validated by qPCR. Results We identified a subset of radiation-modulated genes whose expression was affected by silencing of both TP53 and RELA, and a subset of radiation-upregulated genes where radiation stimulated binding of both p53 and RelA. For three genes, namely IL4I1, SERPINE1, and CDKN1A, an antagonistic effect of the TP53 and RELA silencing was consistent with radiation-enhanced binding of both p53 and RelA. This suggested the possibility of a direct antagonistic (co)regulation by both factors: activation by NF-κB and inhibition by p53 of IL4I1, and activation by p53 and inhibition by NF-κB of CDKN1A and SERPINE1. On the other hand, radiation-enhanced binding of both p53 and RelA was observed in a putative regulatory region of the RRAD gene whose expression was downregulated both by TP53 and RELA silencing, which suggested a possibility of direct (co)activation by both factors. Conclusions Four new candidates for genes directly co-regulated by NF-κB and p53 were revealed. Electronic supplementary material The online version of this article (10.1186/s12864-018-5211-y) contains supplementary material, which is available to authorized users.

The aim of this study was to investigate the cytotoxic, antiproliferative activity and the induction of apoptosis by L-amino acid oxidase isolated from Calloselasma rhodostoma crude venom (CR-LAAO) on human colon cancer cells. CR-LAAO was purified using three chromatographic steps: molecular exclusion using G-50 gel filtration resin, ion-exchange by MonoQ column and desalted on a G25 column. The purity and identity of the isolated CR-LAAO was confirmed by SDS-PAGE and LC-MS/MS. CR-LAAO demonstrated time- and dose-dependent cytotoxic activity on SW480 (primary human colon cancer cells) and SW620 (metastatic human colon cancer cells) with an EC50 values of 6 μg/ml and 7 μg/ml at 48 hr, respectively. Quantification of apoptotic cells based on morphological features demonstrated significant increase in apoptotic cell population in both SW480 and SW620 cells which peaked at 48 hr. Significant increase in caspase-3 activity and reduction in Bcl-2 levels were demonstrated following CR-LAAO treatment. These data provide evidence on the potential anticancer activity of CR-LAAO from the venom of C. rhodostoma for therapeutic intervention of human colon cancer.

Snake venom L-amino acid oxidases (SV-LAAOs) are enzymes of great interest in research due to their many biological effects with therapeutic potential. CR-LAAO, an L-amino acid oxidase from Calloselasma rhodostoma snake venom, is a well described SV-LAAO with immunomodulatory, antiparasitic, microbicidal, and antitumor effects. In this study, we evaluated the genotoxic potential of this enzyme in human peripheral blood mononuclear cells (PBMC) and HepG2 tumor cells, as well as its interaction with these cells, its impact on the expression of DNA repair and antioxidant pathway genes, and reactive oxygen species (ROS)-induced intracellular production. Flow cytometry analysis of FITC-labelled CR-LAAO showed higher specificity of interaction with HepG2 cells than PBMC. Moreover, CR-LAAO significantly increased intracellular levels of ROS only in HepG2 tumor cells, as assessed by fluorescence. CR-LAAO also induced genotoxicity in HepG2 cells and PBMC after 4 h of stimulus, with DNA damages persisting in HepG2 cells after 24 h. To investigate the molecular basis underlying the genotoxicity attributed to CR-LAAO, we analyzed the expression profile (mRNA levels) of 44 genes involved in DNA repair and antioxidant pathways in HepG2 cells by RT2 Profiler polymerase chain reaction array. CR-LAAO altered the tumor cell expression of DNA repair genes, with two downregulated (XRCC4 and TOPBP1) and three upregulated (ERCC6, RAD52 and CDKN1) genes. In addition, two genes of the antioxidant pathway were upregulated (GPX3 and MPO), probably in an attempt to protect tumor cells from oxidative damage. In conclusion, our data suggest that CR-LAAO possesses higher binding affinity to HepG2 tumor cells than to PBMC, its genotoxic mechanism is possibly caused by the oxidative stress related to the production of H2O2, and is also capable of modulating genes related to the DNA repair system and antioxidant pathways.

The present study highlights the cellular mechanism of resistance in human adenocarcinoma (Colo-205) cells against apoptosis induction by Rusvinoxidase, an l -amino acid oxidase purified from Russell's Viper venom (RVV). The significantly lower cytotoxicity as well as apoptotic activity of Rusvinoxidase towards Colo-205 cells (compared to MCF-7 breast cancer cells) is correlated with lower depletion of cellular glutathione content and increased down-regulation of catalase activity of Colo-205 cells following Rusvinoxidase treatment. Exposure to Rusvinoxidase subsequently diminished reactive oxygen species (ROS) production and failed to impair mitochondrial membrane potential, resulting in apoptosis induction resistance in Colo-205 cells. Further, higher expression levels of caspase 8, compared to caspase 9, indicate that Rusvinoxidase preferentially triggers the extrinsic pathway of apoptosis in Colo-205 cells. A time-dependent lower ratio of the relative expression of Bax and Bcl-xL (pro- and anti-apoptotic proteins) in Colo-205 cells, compared to our previous study on MCF-7 cells, unambiguously supports a higher cellular resistance mechanism in Colo-205 cells against Rusvinoxidase-induced apoptosis.

The action of Cr-LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on NADPH oxidase activation in isolated human neutrophil function was investigated. This enzyme has an intrinsic activity of hydrogen peroxide production. Cr-LAAO, in its native form, induces the ROS production in neutrophil and migration of cytosolic NADPH oxidase components p40phox, p47phox and p67phox to the membrane, and Rac, a GTPase protein member, with the involvement of intracellular signaling mediated by phospho PKC-α. In its inactive form, iCr-LAAO does not induce NADPH oxidase activation in neutrophil showing that the intrinsic enzymatic activity does not have a role in this process, suggesting that its primary structure is essential for the cell's stimulation. Accordingly, the data showed for the first time that the Cr-LAAO has a role in NADPH oxidase complex activation triggering relevant proinflammatory events in human neutrophils.

L-amino acid oxidase (LAAO) is a protein toxin commonly found in snake venom. It has many applications, ranging from biotechnology to potential anticancer therapeutics. LAAO converts L-amino acid into α-keto acid and release ammonia and hydrogen peroxide as by-products. Induction of oxidative stress in cancer cells is one of the cancer treatment strategies as controlled and targeted release of hydrogen peroxide can theoretically induce sufficient oxidative stress to kill cancer cells. Furthermore, L-amino acid oxidase has been shown to selectively bind to cell membranes of specific phospholipid composition and deliver the hydrogen peroxide to localized regions on the cell surface. In this mini review, we discuss the relevance of L-amino acid oxidase, in terms of its structure and enzyme activity, its potential as a cytotoxic agent and exploitation of its cytotoxic nature as an anticancer therapeutic.

Ethnopharmacological relevance Bungarus multicinctus snake belongs to Elapidae family and is widely distributed in southern China. It is widely used in traditional Chinese medicine with the effect of dispelling wind and removing obstruction in the meridians. Moreover, it is also as a chief ingredient of many polyherbal formulations for the treatment of cancer. Aim of the study To evaluate the antitumor activity of Bungarus multicinctus snake venom components and isolate, characterize the most effective anti-tumor component of Bungarus multicinctus snake venom. Materials and methods The in vitro antitumor activity of Bungarus multicinctus venom components was detected by cytotoxicity assay and cell apoptosis assay. A unique LAAO from Bungarus multicinctus venom named as BM-Apotxin was isolated and characterized by Sephadex G-75 gel filtration, Sephadex G-25 desalting, Q ion-exchange chromatography and subsequent amino acids sequence determination. The LAAO activity and enzyme kinetics of BM-Apotxin was detected by microplate assay. Results BM-Apotxin, a 65KDa glycoprotein, which contributed to the most anti-tumor effects of Bungarus multicinctus venom. BM-Apotxin can selectively kill tumor cells, with less cytotoxicity to the normal cells. BM-Apotxin is an L-amino acid oxidase (LAAO) with high sequence identity to other snake venom LAAOs. Its anti-tumor activity is mainly due to the hydrogen peroxide produced from LAAO oxidation. But the catalase did not reverse its anti-tumor effect completely. Like other snake venom LAAOs, BM-Apotxin can oxidize many L amino acids, not D amino acids. The optimum substrate for BM-Apotxin is L -Phe. Moreover, BM-Apotxin deglycosylation can significantly reduce the LAAO activity and anti-tumor activity of BM-Apotxin. Conclusion This study will facilitate the study on anti-tumor mechanism of snake venom and drug development based on Bungarus multicinctus venom.

α-Ketoglutaramate (KGM) is an underexamined metabolite of L-glutamine in the metabolic pathway of glutaminase II of α-ketoglutarate formation. Presumably, KGM may be a biomarker of hepatic encephalopathy and other hyperammonemic diseases. This metabolite is a substrate for the ω-amidase enzyme and is used to determine its activity in the study of the biochemistry of various types of cancer. However, the commercial unavailability of KGM hinders its widespread use. Methods for the preparative synthesis of KGM are known, but they either do not provide the proper yield or proper purity of the target product. In this work, a detailed description of the procedures is given that allows the production of KGM with a purity above 97% and a yield of the target product above 75% using L-amino acid oxidase from C. adamanteus as a catalyst of L-glutamine conversion. KGM can be obtained both in the form of a highly concentrated aqueous solution and in the form of crystals of sodium salt. The developed methods can be used both for scaling up the synthesis of KGM and for creating economical biocatalytic technologies for the production of other highly purified preparations.

L-lysine α-oxidase (LO), one of L-amino acid oxidases, deaminates L-lysine with the yield of H2O2, ammonia, and α-keto-ε-aminocaproate. Multiple in vitro and in vivo studies have reported cytotoxic, antitumor, antimetastatic, and antitumor activity of LO. Unlike asparaginase, LO has a dual mechanism of action: depletion of L-lysine and formation of H2O2, both targeting tumor growth. Prominent results were obtained on murine and human tumor models, including human colon cancer xenografts HCT 116, LS174T, and T47D with maximum T/C 12, 37, and 36%, respectively. The data obtained from human cancer xenografts in immunodeficient mice confirm the potential of LO as an agent for colon cancer treatment. In this review, we discuss recently discovered molecular mechanisms of biological action and the potential of LO as anticancer enzyme.

An L-amino acid oxidase from Peruvian Bothrops pictus (Bpic-LAAO) snake venom was purified using a combination of size-exclusion and ion-exchange chromatography. Bpic-LAAO is a homodimeric glycosylated flavoprotein with molecular mass of ∼65 kDa under reducing conditions and ∼132 kDa in its native form as analyzed by SDS-PAGE and gel filtration chromatography, respectively. N-terminal amino acid sequencing showed highly conserved residues in a glutamine-rich motif related to binding substrate. The enzyme exhibited optimal activity towards L-Leu at pH 8.5, and like other reported SV-LAAOs, it is stable until 55 °C. Kinetic studies showed that the cations Ca2+, Mg2+ and Mn2+ did not alter Bpic-LAAO activity; however, Zn2+ is an inhibitor. Some reagents such as β-mercaptoethanol, glutathione and iodoacetate had inhibitory effect on Bpic-LAAO activity, but PMSF, EDTA and glutamic acid did not affect its activity. Regarding the biological activities of Bpic-LAAO, this enzyme induced edema in mice (MED = 7.8 μg), and inhibited human platelet aggregation induced by ADP in a dose-dependent manner and showed antibacterial activity on Gram (+) and Gram (-) bacteria. Bpic-LAAO cDNA of 1494 bp codified a mature protein with 487 amino acid residues comprising a signal peptide of 11 amino acids. Finally, the phylogenetic tree obtained with other sequences of LAAOs, evidenced its similarity to other homologous enzymes, showing two well-established monophyletic groups in Viperidae and Elapidae families. Bpic-LAAO is evolutively close related to LAAOs from B. jararacussu, B. moojeni and B. atrox, and together with the LAAO from B. pauloensis, form a well-defined cluster of the Bothrops genus.

Snake venom L-amino acid oxidase (LAAO) exerts toxicity by inducing hemorrhage, pneumorrhagia, pulmonary edema, cardiac edema, liver cell necrosis etc. Being well conserved, inhibitors of the enzyme may be synthesized using the template of the substrate, substrate binding site and features of the catalytic site of the enzyme. Previous findings showed that aristolochic acid (AA), a major constituent of Aristolochia indica, inhibits Russell's viper venom LAAO enzyme activity since, AA interacts with DNA and causes genotoxicity, derivatives of this compound were synthesized by replacing the nitro group to reduce toxicity while retaining the inhibitory potency. The interactions of AA and its derivatives with LAAO were followed by inhibition kinetics and surface plasmon resonance. Similar interactions with DNA were followed by absorption spectroscopy and atomic force microscopy. LAAO-induced cytotoxicity was evaluated by generation of reactive oxygen species (ROS), cell viability assays, confocal and epifluorescence microscopy. The hydroxyl (AA-OH) and chloro (AA-Cl) derivatives acted as inhibitors of LAAO but did not interact with DNA. The derivatives significantly reduced LAAO-induced ROS generation and cytotoxicity in human embryonic kidney (HEK 293) and hepatoma (HepG2) cell lines. Confocal images indicated that AA, AA-OH and AA-Cl interfered with the binding of LAAO to the cell membrane. AA-OH and AA-Cl significantly inhibited LAAO activity and reduced LAAO-induced cytotoxicity.

A new l-amino acid oxidase (LAAO) from Bothrops jararacussu venom (BjussuLAAO-II) was isolated by using a three-step chromatographic procedure based on molecular exclusion, hydrophobicity, and affinity. BjussuLAAO-II is an acidic enzyme with pI=3.9 and molecular mass=60.36kDa that represents 0.3% of the venom proteins and exhibits high enzymatic activity (4884.53U/mg/mim). We determined part of the primary sequence of BjussuLAAO-II by identifying 96 amino acids, from which 34 compose the N-terminal of the enzyme (ADDRNPLEECFRETDYEEFLEIARNGLSDTDNPK). Multiple alignment of the partial BjussuLAAO-II sequence with LAAOs deposited in the NCBI database revealed high similarity (95-97%) with other LAAOs isolated from Bothrops snake venoms. BjussuLAAO-II exerted a strong antiprotozoal effect against Leishmania amazonensis (IC50=4.56μg/mL) and Trypanosoma cruzi (IC50=4.85μg/mL). This toxin also induced cytotoxicity (IC50=1.80μg/mL) and apoptosis in MCF7 cells (a human breast adenocarcinoma cell line) by activating the intrinsic and extrinsic apoptosis pathways, but were not cytotoxic towards MCF10A cells (a non-tumorigenic human breast epithelial cell line). The results reported herein add important knowledge to the field of Toxinology, especially for the development of new therapeutic agents.

Proteus vulgaris L-amino acid deaminase (pvLAAD) belongs to a class of bacterial membrane-bound LAADs mainly express in genus Proteus, Providencia and Morganella. These LAADs employ a non-cleavable N-terminal twin-arginine translocation (Tat) peptide to transport across membrane and bind to bacterial surface. Recent studies revealed that a hydrophobic insertion sequence (INS) in these LAADs also interacts with bacterial membrane. However, the functional significance of INS-membrane interaction is not clear. In this study, we made site-directed mutagenesis on the surface-exposed hydrophobic residues of pvLAAD INS, and we found that these mutations impaired the INS-membrane interaction but did not affect pvLAAD activity in the solution. We further found that when cell membrane is present, the catalytic activity can be increased by 8~10 folds for wild-type but not INS-mutated pvLAAD, indicating that the INS-membrane interaction is necessary for increasing activity of pvLAAD. Molecular dynamic (MD) simulations suggested that INS is flexible in the solution, and its conformational dynamics could lead to substrate channel distortion. Circular dichroism (CD) spectroscopy experiments indicated that bacterial membrane was able to maintain the conformation of INS. Our study suggests the function of the membrane binding of INS is to stabilize pvLAAD structure and increase its catalytic activity.

Cryptocaryon irritans is an important protozoan parasite which infects almost all kinds of marine teleosts, causing heavy economic losses. In our previous studies, we found that rabbitfish ( Siganus oramin ) displayed high resistance to C. irritans infection, and a novel protein, l -amino acid oxidase (LAAO), was identified from the serum that was lethal to C. irritans . In this study, the rabbitfish were firstly infected with a high dose of C. irritans , then the LAAO mRNA expression pattern and the activity of three enzymes [superoxide dismutase (SOD), Na + /K + -ATPase and Ca 2+ /Mg 2+ -ATPase] were measured in various tissues. The results indicated that, after infection, the feeding and swimming of rabbitfish was normal, and the infection intensity in the host was low. Tissue distribution analysis showed that LAAO mRNA was most pronounced in the head kidney and gill, with lower expression observed in the muscle. After infection with C. irritans , the LAAO mRNA was up-regulated early post infection (from 6 to 24 h) in both gill and spleen, but then returned to normal levels, implying that LAAO may play an important role in the host's early immune response. The SOD activity in the liver was significantly higher in the infection group than in the control group by 48 h post infection, while Na + /K + -ATPase and Ca 2+ /Mg 2+ -ATPase activities in the gill were decreased by 12 and 24 h after infection; no significant difference was detected at the other time points throughout the experiment. Together, these results suggest that biochemical responses of rabbitfish are relatively mild after infection with a high dose of parasite, and the LAAO may play an important role in the host's defense against C. irritans .

Here, two class-enzyme motors are properly designed allowing the rapid dispersion of the class-enzyme D-amino acid oxidase (DAO) and L-amino acid oxidase (LAO) for selective “on the fly” biodetection of D and L-amino acids (AAs), respectively. The efficient movement together with the continuous release of fresh class-enzyme leads to a greatly accelerated enzymatic reaction processes without the need of external stirring or chemical and physical attachment of the enzyme. Ultra-fast detection (

Snake venom enzymes of the L-amino acid oxidase (LAAO) class are responsible for tissue hemorrhage, edema, and derangement of platelet function. However, what role, if any, these flavoenzymes play in altering plasmatic coagulation have not been well defined. Using coagulation kinetomic analyses (thrombelastograph-based), it was determined that the LAAO derived from Crotalus adamanteus venom displayed a procoagulant activity associated with weak clot strength (no factor XIII activation) similar to thrombin-like enzymes. The procoagulant activity was not modified in the presence of reduced glutathione, demonstrating that the procoagulant activity was likely due to deamination, and not hydrogen peroxide generation by the LAAO. Further, unlike the raw venom of the same species, the purified LAAO was not inhibited by carbon monoxide releasing molecule-2 (CORM-2). Lastly, exposure of the enzyme to phenylmethylsulfonyl fluoride (PMSF) resulted in the LAAO expressing anticoagulant activity, preventing contact activation generated thrombin from forming a clot. In sum, this investigation for the first time characterized the LAAO of a snake venom as both a fibrinogen polymerizing and an anticoagulant enzyme acting via oxidative deamination and not proteolysis as is the case with thrombin-like enzymes (e.g., serine proteases). Using this thrombelastographic approach, future investigation of purified enzymes can define their biochemical nature.

Bothrops snake venoms contain biologically active components, including L-amino acid oxidases (LAAO) that induce significant leukocyte accumulation at inflammatory sites characterized by early neutrophil infiltration. As it remains unclear how snake venoms modulate neutrophil activation and chemokine production, here we examined whether Bothrops moojeni crude venom (BmV) and its LAAO (BmooLAAO-I) affect expression of the surface activation markers CD11b and CD66b, production of the chemokines CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8, CXCL9/MIG, and CXCL-10/IP-10, and activation of oxidative burst in human neutrophils. Cell viability, expression of activation markers, and chemokine production were assessed by flow cytometry, while the oxidative burst response was measured by chemiluminescence. BmV at 50 and 75 µg/mL reduced CXCL8/IL-8 (p < 0.001 and p < 0.01, respectively) and CCL2/MCP-1 production (p < 0.05), while BmooLAAO-I at the same concentrations reduced only CCL2/MCP-1 production (p < 0.01). These effects were accompanied by CD11b upregulation (p < 0.05 for 50 and 75 µg/mL BmV; p < 0.01 for 50 and 75 µg/mL BmooLAAO-I) and CD66b downregulation (p < 0.05 for 50 and 75 µg/mL BmV). Both BmV and BmooLAAO-I at concentrations ranging from 0.625 to 5 µg/mL suppressed the oxidative burst of neutrophils stimulated with phorbol 12-myristate 13-acetate, while BmooLAAO-I at 2.5 and 5 µg/mL also suppressed the neutrophil response stimulated with opsonized zymosan. Considering that neutrophils participate in the pathogenesis of autoimmune and inflammatory diseases, the findings reported herein indicate that BmV and BmooLAAO-I are potential immunomodulating agents.

D-danshensu (D-DSS), extracted from the plant Salvia miltiorrhiza (Danshen), is widely used to treat cardiovascular and cerebrovascular diseases. Here we engineered Escherichia coli strains to produce D-DSS from catechol, pyruvate and ammonia by one-pot biotransformation. Tyrosin-phenol lyase (TPL), L-amino acid deaminase (aadL), D-lactate dehydrogenase (ldhD) and glucose dehydrogenase (gdh) genes were overexpressed in Escherichia coli strain. First, the expression of genes was regulated by different copy number plasmids combination, the result of E. coli TALG6, with strong overexpression of TPL, aadL, ldhD and moderate overexpression of gdh, exhibited 253.7% increase D-DSS production compared to E. coli TALG1. Second, the optimum concentration of catechol was found to be 50 mM. Finally, a fed-batch biotransformation strategy was proposed, namely the amount of catechol was added to 50 mM every 2 h. The total production of D-DSS reached 55.35 mM within 14 h, which was 1.7 times that without feeding.

Snake venom L-amino acid oxidases (svLAAOs) are an interesting class of enzymes with important biological activities. Their participation in key metabolic processes, including pathological disorders, suggest that svLAAOs are potential lead compounds in drug discovery. However, their short-term stability defies their applications. This paper describes the stability studies together with functional and structural characterization of the LAAO bordonein-L. It has 498 amino acid residues, one N-glycosylation site and two disulfide bonds, revealed by high-resolution MS/MS. Molecular modeling approach showed its monomer folds into three conserved domains: FAD, substrate and helical domains. Differential scanning fluorimetry showed the enzyme tends to destabilize from neutral to basic pHs and in presence of mono/bivalent ions and it is highly stabilized by acid pHs and its substrates. However, high concentrations of L-amino acids decrease bordonein-L enzyme activity. Dynamic light scattering revealed bordonein-L remains in the dimeric and monodisperse form, so aggregation does not cause the rapidly decrease of enzyme activity. In vitro, the enzyme exhibited cytotoxicity against fibroblast cell line and killed Leishmania amazonensis promastigotes, intensified by substrate addition. Concluding, our results provide biochemistry and biophysical insights to improve LAAOs stability and better approaches to long-term storage. Moreover, our study emphasizes the importance of proper buffers choice mainly in cell-based assays.

l-amino acid oxidase (LAAO) is a recently discovered novel fish immune enzyme. To explore the role of LAAO in the immune system of bony fishes, we cloned the full-length coding sequence (CDS) of LAAO of the zebrafish Danio rerio (ZF-LAAO), conducted bioinformatics analysis of ZF-LAAO, and analyzed its expression profile in zebrafish infected with the pathogen Streptococcus agalactiae. The CDS of ZF-LAAO was 1,515 base pairs long, and the encoded protein of ZF-LAAO contained an 18 amino acid signal peptide. ZF-LAAO contained the conserved domains of the LAAO family (dinucleotide binding motif and GG-motif), 2 N-glycosylation sites, and 2 O-glycosylation sites, and it was a stable hydrophilic exocrine protein. Similarity of the amino acid sequence of ZF-LAAO with LAAOs of 14 other bony fish species was >50% in all cases. The greatest similarity (79.45%) was with the LAAO of Anabarilius grahami, and these two LAAOs were grouped together in the phylogenetic tree. In wild-type zebrafish infected with S. agalactiae, changes in ZF-LAAO gene (zflaao) expression occurred mainly in the early stage of infection, and the changes in zflaao expression were more pronounced than those of the immune enzyme lysozyme (LYZ). The expression levels of both LYZ gene of zebrafish (zflyz) and zflaao were significantly elevated at 6 h after infection (p