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Snake venom L-amino acid oxidases (LAAOs) are flavoenzymes with diverse physiological and pharmacological effects. These enzymes are found to showcase anticoagulant, antiplatelet, cytotoxicity and other biological effects in bite victims. However, the exact mechanism through which they exhibit several biological properties is not yet fully understood. The current study focussed on the purification of cobra venom LAAO and the functional characterization of purified LAAO. A novel L-amino acid oxidase NNLAAO70 with a molecular weight ~70 kDa was purified from the venom of an Indian spectacled cobra (Naja naja). NNLAAO70 showed high substrate specificity for L-His, L-Leu, and L-Arg during its LAAO activity. It inhibited adenosine di-phosphate (ADP) and collagen-induced platelet aggregation process in a dose-dependent manner. About 60% inhibition of collagen-induced and 40% inhibition of ADP-induced platelet aggregation was observed with a 40 μg/ml dose of NNLAAO70. NNLAAO70 exhibited bactericidal activity on Bacillus subtilis, Escherichia coli, Bacillus megaterium, and Pseudomonas fluorescens. NNLAAO70 also showed cytotoxicity on A549 cells in vitro. It showed severe bactericidal activity on P. fluorescens and lysed 55% of cells. NNLAAO70 also exhibited drastic cytotoxicity on A549 cells. At 1 μg/ml dosage, it demonstrated a 60% reduction in A549 viability and induced apoptosis upon 24-h incubation. H2O2 released during oxidative deamination reactions played a major role in NNLAAO70-induced cytotoxicity. NNLAAO70 significantly increased intracellular reactive oxygen species (ROS) levels in A549 cells by six fold when compared to untreated cells. Oxidative stress-mediated cell injury is the primary cause of NNLAAO70-induced apoptosis in A549 cells and prolonged oxidative stress caused DNA fragmentation and activated cellular secondary necrosis. [ABSTRACT FROM AUTHOR]

In this study, we describe the isolation of a new isoform, l-Amino acid oxidase (LAAO), referred to as Balt-LAAO-II, from Bothrops alternatus snake venom, which was highly purified using a combination of molecular exclusion (Sephadex G-75) and RP-HPLC chromatographic steps. SDS-PAGE analysis showed that purified Balt-LAAO-II had a molecular weight of ∼ 66 kDa. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to those of other snake venom l-Amino acid oxidases. This enzyme induces in vitro cytotoxicity in cultured human leukemic HL60 cells. Cells were grown in RPMI medium and incubated with the isoform Balt-LAAO-II (1, 10, and 100 μ g/mL) for up to 72 h. All three concentrations of venom markedly decreased cell viability from 6 h onwards based on staining with propidium iodide, the reduction of 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and the uptake of neutral red. Flow cytometry showed that all isoforms of Balt-LAAO-II and whole venom concentrations induced apoptosis after 2–6 h of incubation. Morphological analysis of cells incubated with the isoform Balt-LAAO-II and whole venom showed cell rounding and lysis, which increased with venom concentration and duration of incubation. These results show that the isoform Balt-LAAO-II from venom Bothrops alternatus is cytotoxic to cultured HL60 cells, suggesting that this damage may involve the apoptotic and oxidative stress pathways. [ABSTRACT FROM AUTHOR]

Biomedical studies of the role of organic selenium compounds indicate that the amino acid derivative of L-selenomethionine, α-ketomethylselenobutyrate (KMSB), can be considered a potential anticancer therapeutic agent. It was noted that, in addition to a direct effect on redox signaling molecules, α-ketoacid metabolites of organoselenium compounds are able to change the status of histone acetylation and suppress the activity of histone deacetylases in cancer cells. However, the wide use of KMSB in biomedical research is hindered not only by its commercial unavailability, but also by the fact that there is no detailed information in the literature on possible methods for the synthesis of this compound. This paper describes in detail the procedure for obtaining a high-purity KMSB preparation (purity ≥ 99.3%) with a yield of the target product of more than 67%. L-amino acid oxidase obtained from C. adamanteus was used as a catalyst for the conversion of L-selenomethionine to KMSB. If necessary, this method can be used as a basis both for scaling up the synthesis of KMSB and for developing cost-effective biocatalytic technologies for obtaining other highly purified drugs. [ABSTRACT FROM AUTHOR]

Snake venom l-amino acid oxidases (svLAAOs) have been recognized as promising candidates for anticancer therapeutics. However, multiple aspects of their catalytic mechanism and the overall responses of cancer cells to these redox enzymes remain ambiguous. Here, we present an analysis of the phylogenetic relationships and active site-related residues among svLAAOs and reveal that the previously proposed critical catalytic residue His 223 is highly conserved in the viperid but not the elapid svLAAO clade. To gain further insight into the action mechanism of the elapid svLAAOs, we purify and characterize the structural, biochemical, and anticancer therapeutic potentials of the Thailand elapid snake Naja kaouthia LAAO (NK-LAAO). We find that NK-LAAO, with Ser 223, exhibits high catalytic activity toward hydrophobic l-amino acid substrates. Moreover, NK-LAAO induces substantial oxidative stress-mediated cytotoxicity with the magnitude relying on both the levels of extracellular hydrogen peroxide (H2O2) and intracellular reactive oxygen species (ROS) generated during the enzymatic redox reactions, but not being influenced by the N-linked glycans on its surface. Unexpectedly, we discover a tolerant mechanism deployed by cancer cells to dampen the anticancer activities of NK-LAAO. NK-LAAO treatment amplifies interleukin (IL)-6 expression via the pannexin 1 (Panx1)-directed intracellular calcium (iCa2+) signaling pathway to confer adaptive and aggressive phenotypes on cancer cells. Accordingly, IL-6 silencing renders cancer cells vulnerable to NK-LAAO-induced oxidative stress together with abrogating NK-LAAO-stimulated metastatic acquisition. Collectively, our study urges caution when using svLAAOs in cancer treatment and identifies the Panx1/iCa2+/IL-6 axis as a therapeutic target for improving the effectiveness of svLAAOs-based anticancer therapies.

The mere glimpse of venomous animals has always terrified humans because of the devastating effects of their venoms. However, researchers across the globe have isolated therapeutically active ingredients from these venoms and continue to explore them for drug leads. These efforts lead to the discovery of therapeutic molecules that the USFDA has approved to treat different diseases, such as hypertension (Captopril), chronic pain (Ziconotide), and diabetes (Exenatide). The main active constituents of most venoms are proteins and peptides, which gained more attention because of advancements in biotechnology and drug delivery. The utilization of newer screening approaches improved our understanding of the pharmacological complexity of venom constituents and facilitated the development of novel therapeutics. Currently, with many venom-derived peptides undergoing different phases of clinical trials, more are in pre-clinical drug development phases. This review highlights the various sources of venoms, their pharmacological actions, and the current developments in venom-based therapeutics. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T-cell therapy plays a critical role in the treatment of B-cell hematologic malignancies. The combination of PD-1 inhibitors and CAR-T has shown encouraging results in treating patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). However, there are still cases where treatment is ineffective. This study aimed to investigate the role of IL4I1 in the poor efficacy of CD19 CAR-T combined with PD-1 inhibitors in R/R DLBCL and to explore potential mechanisms. Transcriptomic and metabolomic correlation analyses were performed on tumor tissue from DLBCL patients. We employed an in vitro co-culture system consisting of Pfeiffer cells, CD19 CAR-T and macrophages to investigate the underlying mechanisms. It was found that IL4I1 levels were significantly increased in the tumor tissues of R/R DLBCL patients compared to responders. Correlation analysis revealed a positive association between IL4I1 and tryptophan (Trp)-kynurenic acid (Kyn) related metabolites. In the in vitro co-culture model, the presence of IL4I1 inhibited the cytotoxicity of CAR-T cells. Depletion of IL4I1 disrupted the IDO-AHR-Kyn signaling pathway, thereby enhancing the effectiveness of PD-1 inhibitors in combination with CD19 CAR-T for DLBCL treatment. CAR-T-mediated cytotoxicity was significantly inhibited when IL4I1 was present in the in vitro co-culture model. These findings suggest that IL4I1 may be a contributing factor to poor prognosis in R/R DLBCL patients. IL4I1 expression enhances immunosuppression via the IDO-AHR-Kyn pathway, inhibiting the effectiveness of PD-1 inhibitors combined with CD19 CAR-T. Therefore, suppression of IL4I1 may represent a potential target for combination therapy in DLBCL., Competing Interests: Declarations. Ethics approval and consent to participate: Informed consent was provided with lymphoma and healthy donors agreed to participate in this experiment within a clinical trial. Ethics approval was provided at the Department of Hematology at Tianjin First Central Hospital (Tianjin, China) (ChiCTR-ONN-16009862; Tianjin First Central Hospital Medical Ethics Committee). Competing interest: The authors declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article., (© 2024. The Author(s).)

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 grouper 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, CaCl 2 , MgCl 2 , and diluted seawater. The activity was non-linearly increased and fitted to the four-parameter log-logistic model. The EC 50 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. The results of circular dichroism spectroscopy 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 enzymatic activity of grouper serum LAO was regulated. • In the grouper body, LAO was inactive to reduce the toxicity of catalytic byproducts. • The cations randomized the structure of the LAO to active form. • It was suggested that LAO was activated by mixing with seawater after leaking from the wound. • Grouper serum LAO could play a role in infection control at the wound site. [ABSTRACT FROM AUTHOR]

L‐amino acid oxidases (LAAOs) with broad substrate specificity can be used in the deracemization of D,L‐amino acids (D,L‐AAs) to their D‐enantiomers. Hyper‐thermostable LAAO (HTAncLAAO) was designed through a combination of manual sequence data mining and ancestral sequence reconstruction. Soluble expression of HTAncLAAO (>50 mg/L) can be achieved using an E. coli system. HTAncLAAO, which recognizes seven L‐AAs as substrates, exhibits extremely high thermal stability and long‐term stability; the t1/2 value was 95 °C and <5% activity loss after incubation of the enzyme at 30 °C for 1 week. Deracemization could be achieved at 40 °C with a small amount of enzymes compared to previously reported LAAOs. A total of 0.4 mg (2 U) of HTAncLAAO is enough to deracemize three D,L‐AAs at a preparative scale with high enantiomeric excess (>99 % ee, D‐enantiomer). These results suggest that HTAncLAAO is an excellent biocatalyst to perform this deracemization. [ABSTRACT FROM AUTHOR]

One of the deadliest enzymes in the snake venom is l-amino acid oxidase (LAAO) which plays an important role in the pathophysiological effects during snake envenomation. Some effects of this enzyme on the human body are apoptosis, platelet aggregation, edema, hemorrhage, and cytotoxicity. Hence, inhibiting the enzyme activity to reduce its degradation effects is of great medical and pharmacological importance. On the other hand, drug repurposing is a process to find the new existing drug for a new medical indication. Since Crotalus adamanteus LAAO has no crystal structure in the protein data bank, first, its 3D structure was constructed by homology modeling using 1REO as the template and then modeled structure was evaluated by several algorithms. We screened the FDA-approved drugs by structure-based virtual screening, molecular dynamics (MD) simulation, and Molecular Mechanics Poisson Boltzmann Surface Area (MM/PBSA) to identify new inhibitors for the snake venom LAAO. Interestingly, docking results revealed that half of the hits belong to the propionic acid derivatives drugs. In addition, MD simulation was performed to assess the interaction profile of the docked protein–hits complexes. Meanwhile, Arg88, Gln112, Lys345, Trp356 form consistent hydrogen bond interactions with Dexketoprofen, Flurbiprofen, Ketoprofen, Morphine, and Citric acid during simulation. According to the results, each of the four compounds can be an appropriate inhibitor of LAAO and since our study was based on drug repurposing could be evaluated in phase II clinical trials. [ABSTRACT FROM AUTHOR]

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.

Abstract 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.

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. Key points: •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. [ABSTRACT FROM AUTHOR]

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. Image 1 • Crotalus durissus terrificus venom plasticity. • Difference between snake venoms from different environments (Captivity - Nature). • L-amino acid oxidase purification of Crotalus durissus terrificus venom. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Despite the immune system's role in the detection and eradication of abnormal cells, cancer cells often evade elimination by exploitation of various immune escape mechanisms. Among these mechanisms is the ability of cancer cells to upregulate amino acid-metabolizing enzymes, or to induce these enzymes in tumor-infiltrating immunosuppressive cells. Amino acids are fundamental cellular nutrients required for a variety of physiological processes, and their inadequacy can severely impact immune cell function. Amino acid-derived metabolites can additionally dampen the anti-tumor immune response by means of their immunosuppressive activities, whilst some can also promote tumor growth directly. Based on their evident role in tumor immune escape, the amino acid-metabolizing enzymes glutaminase 1 (GLS1), arginase 1 (ARG1), inducible nitric oxide synthase (iNOS), indoleamine 2,3-dioxygenase 1 (IDO1), tryptophan 2,3-dioxygenase (TDO) and interleukin 4 induced 1 (IL4I1) each serve as a promising target for immunotherapeutic intervention. This review summarizes and discusses the involvement of these enzymes in cancer, their effect on the anti-tumor immune response and the recent progress made in the preclinical and clinical evaluation of inhibitors targeting these enzymes., Competing Interests: Author YG was employed by Oncolines B.V., (Copyright © 2024 Grobben.)

Successful implementation of enzymes in practical application hinges on the development of efficient mass production techniques. However, in a heterologous expression system, the protein is often unable to fold correctly and, thus, forms inclusion bodies, resulting in the loss of its original activity. In this study, we present a new and more accurate model for predicting amino acids associated with an increased L-amino acid oxidase (LAO) solubility. Expressing LAO from Rhizoctonia solani in Escherichia coli and combining random mutagenesis and statistical logistic regression, we modified 108 amino acid residues by substituting hydrophobic amino acids with serine and hydrophilic amino acids with alanine. Our results indicated that specific mutations in Euclidean distance, glycine, methionine, and secondary structure increased LAO expression. Furthermore, repeated mutations were performed for LAO based on logistic regression models. The mutated LAO displayed a significantly increased solubility, with the 6-point and 58-point mutants showing a 2.64- and 4.22-fold increase, respectively, compared with WT-LAO. Ultimately, using recombinant LAO in the biotransformation of α-keto acids indicates its great potential as a biocatalyst in industrial production., (© 2024 Wiley-VCH GmbH.)

In recent years, a novel treatment method for cancer has emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the l-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata . Previously isolated from the ink, the l-amino acid oxidase was described to oxidate the essential amino acids l-lysine and l-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of the amino acid oxidase Aplysia punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose-dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG, which led to deprivation of arginine, was well tolerated.

The pathophysiological effects of Russell's viper venom (RVV) and its fractions, including phospholipase A2 (RvPLA2), metalloprotease (RvMP), L-amino acid oxidase (RvLAAO), and phosphodiesterase (RvPDE) on renal functions were investigated using the isolated perfused rabbit kidney (IPK) model. Moreover, whether their effects on renal alterations were promoted by platelet activating factor (PAF) was tested using the PAF receptor antagonist, WEB 2086. There was a marked reduction in the perfusion pressure (PP) and renal vascular resistance (RVR) 10 min after RVV administration (1.0 mg/100 ml of perfusate), thereafter both PP and RVR gradually increased and approached the control level within 90 min. These effects were abolished by pretreatment with WEB2086 (2 μg/μl). Administration with RvPLA2 (280 μg/ml), RvMP (280 μg/ml), or RvLAAO (135 μg/ml) alone increased both the PP and RVR, whereas RvPDE (100 μg/ml) reduced both the PP and RVR. Pretreatment with WEB 2086 completely abolished the effects induced by RvMP, but not the other fractions. The RVV also caused a marked decrease in the glomerular filtration rate (GFR), urinary flow rate (UF), and osmolar clearance (Cosm), and these effects were not inhibited by pretreatment with WEB2086. Each RVV fraction also increased, to varying extents, the GFR, UF, and Cosm, and these effects induced by RvPLA2 or RvMP, but not the other fractions, were completely blocked by WEB 2086. Changes in percent filtered Na+ and K+ excreted in the IPK by RVV, RvPDE, and RvMP were abolished by pretreatment with WEB 2086. Histological evaluation profiled mainly tubulonephrosis in the treated kidney. These results reveal that the alterations in renal functions induced by RVV and its fractions are due to the synergistic action of the different components of snake venom, instead of the action of a single component. The effects of RVV and its fractions in rabbit IPK are mediated at least in part by PAF.

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.

Abstract Background L-amino acid oxidases isolated from snake venoms (SV-LAAOs) are enzymes that have great therapeutic potential and are currently being investigated as tools for developing new strategies to treat various diseases, including cancer and bacterial infections. The main objective of this study was to make a brief evaluation of the enzymatic stability of two Bothrops LAAOs, one isolated from Bothrops jararacussu (BjussuLAAO-II) and the other from Bothrops moojeni (BmooLAAO-I) venoms. Methods and results The enzymatic activity and stability of both LAAOs were evaluated by microplate colorimetric assays, for which BjussuLAAO-II and BmooLAAO-I were incubated with different L-amino acid substrates, in the presence of different ions, and at different pH ranges and temperatures. BjussuLAAO-II and BmooLAAO-I demonstrated higher affinity for hydrophobic amino acids, such as Phe and Leu. The two enzymes showed high enzymatic activity in a wide temperature range, from 25 to 75 °C, and presented optimum pH around 7.0. Additionally, Zn2+, Al3+, Cu2+ and Ni2+ ions negatively modulated the enzymatic activity of both LAAOs. As to stability, BjussuLAAO-II and BmooLAAO-I showed high enzymatic activity for 42 days stored at 4 °C in neutral pH solution. Moreover, the glycan portions of both LAAOs were analyzed by capillary electrophoresis, which revealed that BjussuLAAO-II presented two main glycan portions with relative masses of 7.78 and 8.13 CGU, while BmooLAAO-I showed three portions of 7.58, 7.94 and 8.37 CGU. Conclusions Our results showed that, when stored properly, BjussuLAAO-II and BmooLAAO-I present enzymatic stability over a long time period, which is very important to allow the use of these enzymes in pharmacological studies of great impact in the medical field.

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. • BmooLAAO-II is a new SV-LAAO isoform that we isolated from Bothrops moojeni venom • BjussuLAAO-II and BmooLAAO-II are strongly cytotoxic to two Leishmania species • Both SV-LAAOs have antiproliferative action and alter the mitochondrial potential • Each SV-LAAO induces ROS production in only one of the Leishmania species [ABSTRACT FROM AUTHOR]

L-lysine α-oxidase (LO) is an L-amino acid oxidase with antitumor, antimicrobial and antiviral properties. Pharmacokinetic (PK) studies were carried out by measuring LO concentration in plasma and tissue samples by enzyme immunoassay. L-lysine concentration in samples was measured spectrophotometrically using LO. After single i.v. injection of 1.0, 1.5, 3.0 mg/kg the circulating T1/2 of enzyme in mice varied from 51 to 74 min and the AUC0–inf values were 6.54 ± 0.46, 8.66 ± 0.59, 9.47 ± 1.45 μg/ml × h, respectively. LO was distributed in tissues and determined within 48 h after administration with maximal accumulation in liver and heart tissues. Mean time to reach the maximum concentration was highest for the liver—9 h, kidney—1 h and 15 min for the tissues of heart, spleen and brain. T1/2 of LO in tissues ranged from 7.75 ± 0.73 to 26.10 ± 2.60 h. In mice, plasma L-lysine decreased by 79% 15 min after LO administration in dose 1.6 mg/kg. The serum L-lysine levels remained very low from 1 to 9 h (< 25 μM, 17%), indicating an acute lack of L-lysine in animals for at least 9 h. Concentration of L-lysine in serum restored only 24 h after LO administration. The results of LO PK study show that it might be considered as a promising enzyme for further investigation as a potential anticancer agent. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

• Oxidation of an l -amino acid using a recombinant l -amino acid oxidase from Hebeloma cylindrosporum was studied. • Properties of this enzyme were determined for the formation of phenylpyruvic acid from l -phenylalanine as a model reaction. • The enzyme displayed a reasonable operational stability in the reaction system. • Promising applicability data with respect to substrate and product inhibition were found. • In a biotransformation, 20 mM of substrate were converted after 4 h reaction. The biocatalytic oxidation of amino acids represents an attractive approach towards the synthesis of α-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 α-keto 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, 20 mM of substrate were converted after 4 h reaction. The formation of undesired by-products was suppressed using a commercially available catalase enzyme. [ABSTRACT FROM AUTHOR]

• l -amino acid oxidase from the venom of M. lemniscatus snake (ML-LAAO) was characterized • ML-LAAO is a glycoprotein, optimal activity at pH 8.5 and specificity to hydrophobic l -amino acids • ML-LAAO have blocked weakly aggregation induced by collagen on washed platelets • ML-LAAO displayed antibacterial activity against Staphylococcus aureus. • ML-LAAO displayed leishmanial action against Leishmania amazonensis and L. chagasi This study reports the purification of ML-LAAO, a new LAAO from the venom of Micrurus lemniscatus snake (ML-V), using size exclusion chromatography. ML-LAAO is a 69-kDa glycoprotein that represents ~2.0% of total venom proteins. This enzyme exhibited optimal activity at pH 8.5, displaying high specificity toward hydrophobic l -amino acids. MALDI TOF/TOF and Blast analysis identified internal segments in ML-LAAO that share high sequence identity with homologous snake venom LAAOs. Western blot analysis on two-dimensional SDS-PAGE of ML-V, using anti-LAAO revealed the presence of ML-LAAO isoforms (pI 6.3–8.9). ML-LAAO blocked aggregation induced by collagen on washed platelets in a rather weak manner, it did not, however, inhibit platelet aggregation induced by ADP on platelet-rich plasma. In addition, this enzyme displayed in vitro antibacterial activity against Staphylococcus aureus (MIC/MBC of 0.39 μg/mL) and in vitro leishmanicidal action against Leishmania amazonensis and L. chagasi (IC 50 values of 0.14 and 0.039 μg/mL, respectively). These activities were significantly reduced by catalase, suggesting that hydrogen peroxide production is involved in some way. The data presented here revealed that ML-LAAO has bactericidal and leishmanicidal effects, suggesting that it may have therapeutic potential. [ABSTRACT FROM AUTHOR]

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 (Ap LAO) and Infundibulicybe geotropa (Cg LAO). 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 (Cg mycLAO). 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 Ap LAO, Cg LAO, and Cg mycLAO. 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 Cg LAO and Cg mycLAO are confirmed in vivo in tomato plants, while Ap LAO 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. [ABSTRACT FROM AUTHOR]

Siganus oramin l -amino acid oxidase (SR-LAAO), isolated from the serum of Siganus oramin , is an innate immune protein with significant antibacterial activity. The aim of this study was to express SR-LAAO in insect cells using a baculovirus expression system and evaluate the function of the recombinant SR-LAAO. To this end, an optimized sequence of the SR-LAAO gene was designed and synthesized, based on the codon bias of insect cells. Bacmid shuttle vectors and recombinant baculovirus were successfully constructed, and the recombinant baculovirus was transfected into Sf9 insect cells. The antibacterial activity and enzymatic characteristics of the recombinant SR-LAAO were investigated. The results showed that the pFastBac-optiSR-LAAO shuttle vectors and Bacmid-optiSR-LAAO were correctly constructed. The Sf9 insect cells exhibited significant cytopathic effects following infection with Bacmid-optiSR-LAAO and Bacmid; the specific PCR analysis proved that the recombinant baculovirus was successfully constructed. The immunofluorescence assay revealed that the recombinant baculovirus rSR-LAAO was abundantly expressed in infected Sf9 insect cells; the results of SDS/PAGE and Western blot analyses showed that a specific band appeared at about 60 kDa. Moreover, the crude rSR-LAAO enzyme displayed strong antibacterial activity against aquatic pathogens, particularly Streptococcus agalactiae and Streptococcus iniae. In addition, the results of catalase interference test implied that the antibacterial activity of rSR-LAAO was directly associated with (H 2 O 2 production). The results of the rSR-LAAO enzymatic characteristics test indicated that the Km value with l -Lysine as a substrate was 16.61 mM when the temperature was under 37 °C, and the optimum pH was 7. The antibacterial activity of rSR-LAAO could be completely inhibited by 10 mg/mL of pepsin, trypsin, and proteinase K compared with both methanol and acetone. Adding an equal volume of ethanol had a minimal impact on the antibacterial activity of rSR-LAAO. The crude enzyme could maintain a high level of antibacterial activity against both Gram-positive and -negative bacteria from 4 °C to 30 °C. In the present study, SR-LAAO was successfully expressed in Sf9 cells using the baculovirus expression system, and provides basic references for further research into the role of LAAO in marine animals and the development of new antimicrobial drugs. • SR-LAAO was successfully recombinantly expressed in insect cells. • The rSR-LAAO displayed strong antibacterial activity against common aquatic pathogens. • Antibacterial activity of rSR-LAAO was directly associated with H 2 O 2. [ABSTRACT FROM AUTHOR]

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.

Enantiomerically pure D-amino acids hold significant potential as precursors for synthesizing various fine chemicals, including peptide-based drugs and other pharmaceuticals. This study focuses on establishing an enzymatic cascade system capable of converting various L-amino acids into their D-isomers. The system integrates four enzymes: ancestral L-amino acid oxidase (AncLAAO-N4), D-amino acid dehydrogenase (DAADH), D-glucose dehydrogenase (GDH), and catalase. AncLAAO-N4 initiates the process by converting L-amino acids to corresponding keto acids, which are then stereo-selectively aminated to D-amino acids by DAADH using NADPH and NH 4 Cl. Concurrently, any generated H 2 O 2 is decomposed into O 2 and H 2 O by catalase, while GDH regenerates NADPH from D-glucose. Optimization of reaction conditions and substrate concentrations enabled the successful synthesis of five D-amino acids, including a D-Phe derivative, three D-Trp derivatives, and D-phenylglycine, all with high enantiopurity (>99 % ee) at a preparative scale (>100 mg). This system demonstrates a versatile approach for producing a diverse array of D-amino acids., (© 2024 Wiley‐VCH GmbH.)

L-amino acid oxidase (AAO) was reported to be capable of converting L-glutamic acid to α-aketoglutaric acid (α-KG). The sequence of AAO from Kitasatospora cheerisanensis was synthesized based on Pichia pastoris codon-usage preferences. AAO gene was cloned into plasmid pPICZα which was transformed into P. pastoris. Next, multi-copy expression plasmids were constructed by using plasmid pHBM905BDM. High-density fermentation was performed and the recombinant enzyme was characterized. The conversion conditions were optimized. By using Escherichia coli expression system, no soluble or active AAO was obtained from two strains after fermentation and induction. We can't obtain high-level expression of recombinant strains by using plasmid pPICZα. Therefore, we constructed multi-copy expression plasmids using plasmid pHBM905BDM. By using this plasmid, multi-copy strains were constructed and named as PAAO1, PAAO2, PAAO3, PAAO4, and PAAO5, respectively. The following results showed that expression of AAO in multicopy strains increased as designed and strain PAAO5 was chosen for high-density fermentation and enzyme activity experiments. After high-density fermentation, we achieved an AAO-expression yield of 120.8 U/mL. After temperature and pH optimization, the highest AAO activity was observed at a temperature and pH of 20°C and 6, respectively. After optimization of the conversion conditions, the average production rate of L-glutamic acid to α-KG was 3.46 g/L/h and the highest α-KG titer (103 g/L) was converted from 120 g/L L-glutamic acid. In this study, AAO was abundantly expressed by using P. pastoris expression system. The following experiments indicated that AAO is suitable for use in industrial applications. [ABSTRACT FROM AUTHOR]

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 H 2 O 2 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 H 2 O 2 -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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ABSTRACT BACKGROUND: Myeloproliferative neoplasms are Philadelphia chromosome-negative diseases characterized by hyperproliferation of mature myeloid cells, associated or not with the Janus kinase 2 tyrosine kinase mutation, JAK2V617F. As there is no curative therapy, researchers have been investigating new drugs to treat myeloproliferative neoplasms, including l-amino acid oxidase from Calloselasma rhodostoma snake venom (CR-LAAO), which is a toxin capable of eliciting apoptosis in several tumor cell lines. OBJECTIVE: To evaluate the effects of l-amino acid oxidase from C. rhodostoma snake venom in the apoptotic machinery of JAK2-mutated cell lines. METHODS: The HEL 92.1.7 and SET-2 cell lines were cultured with l-amino acid oxidase and catalase for 12 h at 37 °C in 5% carbon dioxide. The cell viability was assessed by the multi-table tournament method, the level of apoptosis was measured by flow cytometry, and the expression of cysteine-dependent aspartate-specific proteases and cleaved Poly(ADP-ribose) polymerase were analyzed by Western blotting. RESULTS: l-Amino acid oxidase from C. rhodostoma snake venom was cytotoxic to HEL 92.1.7 and SET-2 cells (50% inhibitory concentration = 0.15 µg/mL and 1.5 µg/mL, respectively) and induced apoptosis in a concentration-dependent manner. Cell treatment with catalase mitigated the l-amino acid oxidase toxicity, indicating that hydrogen peroxide is a key component of its cytotoxic effect.The activated caspases 3 and 8 expression and cleaved PARP in HEL 92.1.7 and SET-2 cells confirmed the apoptosis activation by CR-LAAO. CONCLUSIONS: l-Amino acid oxidase from C. rhodostoma snake venom is a potential antineoplastic agent against HEL 92.1.7 and SET-2 JAK2V617F-positive cells as it activates the extrinsic apoptosis pathway.

Pergularia daemia (Forsk.) chiov., is a milk weed of Asclepiadaceae family. In the present study β-sitosterol, β-amyrin, α-amyrin and lupeol were identified in the leaf by GC–MS. Molecular docking studies were performed to evaluate their activities on phospholipase A2 (PLA2) and l-amino acid oxidase enzymes which constituted a rich source in snake venoms (Naja naja). Snake venom Phospholipase A2 with PDB code 1A3D devoid of co-crystallized ligand was extracted from Protein Data Bank. Using Molegro Virtual Docker two cavities are formed by cocrystallization. l-Amino acid oxidase (PDB code 4E0V) was a receptor model with a co-crystallized ligand FAD. Among the phytochemicals analysed, β-sitosterol displayed high affinity of binding to the active site regions of phospholipase A2 and l-amino acid oxidase, respectively. The affinity of binding was −125.939 and −157.521 kcal/mole identified by gold scores. α-Amyrin and β-amyrin had two hydrogen bond interactions with PLA2. Hence this study suggests that β-sitosterol identified in P. daemia can antagonize PLA2 and LAAO activities and forms a theoretical basis for the folk use of the plant against snake venom.

L-amino acid oxidases (LAAOs) are ubiquitous enzymes in nature. Bioactivities described for these enzymes include apoptosis induction, edema formation, induction or inhibition of platelet aggregation, as well as antiviral, antiparasite, and antibacterial actions. With over 80 species, Micrurus snakes are the representatives of the Elapidae family in the New World. Although LAAOs in Micrurus venoms have been predicted by venom gland transcriptomic studies and detected in proteomic studies, no enzymes of this kind have been previously purified from their venoms. Earlier proteomic studies revealed that the venom of M. mipartitus from Colombia contains ∼4% of LAAO. This enzyme, here named MipLAAO, was isolated and biochemically and functionally characterized. The enzyme is found in monomeric form, with an isotope-averaged molecular mass of 59,100.6 Da, as determined by MALDI-TOF. Its oxidase activity shows substrate preference for hydrophobic amino acids, being optimal at pH 8.0. By nucleotide sequencing of venom gland cDNA of mRNA transcripts obtained from a single snake, six isoforms of MipLAAO with minor variations among them were retrieved. The deduced sequences present a mature chain of 483 amino acids, with a predicted pI of 8.9, and theoretical masses between 55,010.9 and 55,121.0 Da. The difference with experimentally observed mass is likely due to glycosylation, in agreement with the finding of three putative N-glycosylation sites in its amino acid sequence. A phylogenetic analysis of MmipLAAO placed this new enzyme within the clade of homologous proteins from elapid snakes, characterized by the conserved Serine at position 223, in contrast to LAAOs from viperids. MmipLAAO showed a potent bactericidal effect on S. aureus (MIC: 2 µg/mL), but not on E. coli. The former activity could be of interest to future studies assessing its potential as antimicrobial agent.

Background: Glioma is the prevailing malignant intracranial tumor, characterized by an abundance of macrophages. Specifically, the infiltrating macrophages often display the M2 subtype and are known as tumor-associated macrophages (TAMs). They have a critical role in promoting the oncogenic properties of tumor cells. Interleukin-4-induced-1 (IL4I1) functions as an L-phenylalanine oxidase, playing a key part in regulating immune responses and the progression of various tumors. However, there is limited understanding of the IL4I1-mediated cross-talk function between TAMs and glioma cell in the glioma microenvironment., Methods: TCGA, GTEx, and HPA databases were applied to assess the IL4I1 expression, clinical characteristics, and prognostic value of pan-cancer. The link between IL4I1 levels and the prognosis, methylation, and immune checkpoints (ICs) in gliomas were explored through Kaplan-Meier curve, Cox regression, and Spearman correlation analyses. The IL4I1 levels and their distribution were investigated by single-cell analysis and the TIMER 2 database. Additionally, validation of IL4I1 expression was performed by WB, RT-qPCR, IHC, and IF. Co-culture models between glioma cells and M2-like macrophages were used to explore the IL4I1-mediated effects on tumor growth, invasion, and migration of glioma cells. Moreover, the function of IL4I1 on macrophage polarization was evaluated by ELISA, RT-qPCR, WB, and siRNA transfection., Results: Both transcriptome and protein levels of IL4I1 were increased obviously in various tumor types, and correlated with a dismal prognosis. Specifically, IL4I1 was implicated in aggressive progression and a dismal prognosis for patients with glioma. A negative association was noticed between the glioma grade and DNA promoter methylation of IL4I1. Enrichment analyses in glioma patients suggested that IL4I1 was linked to cytokine and immune responses, and was positively correlated with ICs. Single-cell analysis, molecular experiments, and in vitro assays showed that IL4I1 was significantly expressed in TAMs. Importantly, co-culture models proved that IL4I1 significantly promoted the invasion and migration of glioma cells, and induced the polarization of M2-like macrophages., Conclusion: IL4I1 could be a promising immunotherapy target for selective modulation of TAMs and stands as a novel macrophage-related prognostic biomarker in glioma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ye, Wang, Li, Dong, Zhou and Xu.)

Two l-amino acid oxidase enzyme isoforms, Cc-LAAOI and Cc-LAAOII were purified to apparent homogeneity from Cerastes cerastes venom in a sequential two-step chromatographic protocol including; gel filtration and anion exchange chromatography. The native molecular weights of the isoforms were 115 kDa as determined by gel filtration on calibrated Sephacryl S-200 column, while the monomeric molecular weights of the enzymes were, 60, 56 kDa and 60, 53 kDa for LAAOI and LAAOII, respectively. The tryptic peptides of the two isoforms share high sequence homology with other snake venom l-amino acid oxidases. The optimal pH and temperature values of Cc-LAAOI and Cc-LAAOII were 7.8, 50 °C and 7, 60 °C, respectively. The two isoenzymes were thermally stable up to 70 °C. The Km and Vmax values were 0.67 mM, 0.135 μmol/min for LAAOI and 0.82 mM, 0.087 μmol/min for LAAOII. Both isoenzymes displayed high catalytic preference to long-chain, hydrophobic and aromatic amino acids. The Mn2+ ion markedly increased the LAAO activity for both purified isoforms, while Na+, K+, Ca2+, Mg2+ and Ba2+ ions showed a non-significant increase in the enzymatic activity of both isoforms. Furthermore, Zn2+, Ni2+, Co2+, Cu2+ and AL3+ ions markedly inhibited the LAAOI and LAAOII activities. l-Cysteine and reduced glutathione completely inhibited the LAAO activity of both isoenzymes, whereas, β-mercaptoethanol, O-phenanthroline and PMSF completely inhibited the enzymatic activity of LAAOII. Furthermore, iodoacitic acid inhibited the enzymatic activity of LAAOII by 46% and had no effect on the LAAOI activity.

Aim: As the second most prevalent subtype of epithelial ovarian cancers, ovarian clear cell carcinoma (OCCC) is known for its chemoresistance to conventional platinum-based therapy. In this work, we examined the tryptophan (Trp) metabolism enzymes' differential expression in patients with OCCC to assess the potential for personalised treatment., Methods: A total of 127 OCCC tissues were used to construct tissue microarrays, and immunohistochemistry (IHC) staining of the Trp enzymes IDO1, IDO2, TDO2 and IL4I1 was performed. The correlations between Trp enzyme expression and clinical characteristics were analysed., Results: Positive IDO1, IDO2, TDO2 and IL4I1 staining was identified in 26.8%, 94.5%, 75.6% and 82.7% of OCCC respectively. IDO1-positive samples were more common in the chemoresistant group than in the platinum-sensitive group (46.7% vs. 19.8%). Moreover, positive expression of IDO1, TDO2 and IL4I1 was related to advanced stage, metastasis, bilateral tumours, endometriosis and tumour rupture (p < 0.05) respectively. Univariate analysis revealed a significant association between bilateral tumours, lymph node metastasis, advanced stage, distant metastasis and aberrant cytology with a poor prognosis for OCCC, while the absence of residual tumour was correlated with a favourable outcome (p < 0.05). However, only bilateral tumours and lymph node metastases were related to a poor prognosis after multivariate analysis., Conclusion: This is the first study to investigate the expression of the Trp enzymes IDO1, IDO2, TDO2 and IL4I1 in OCCC tissues. IDO2, TDO2 and IL4I1 were detected in the majority of OCCC. Clinical traits were correlated with IDO1, IDO2, TDO2 and IL4I1 expression. IDO1 may be used as a therapeutic target given the large percentage of chemoresistant cases with IDO1 expression. These results will aid the development of personalised therapies for OCCC., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

We describe a new labeling method that allows for full protonation at the backbone Hα position, maintaining protein side chains with a high level of deuteration. We refer to the method as alpha proton exchange by transamination (α-PET) since it relies on transaminase activity demonstrated here using Escherichia coli expression. We show that α-PET labeling is particularly useful in improving structural characterization of solid proteins by introduction of an additional proton reporter, while eliminating many strong dipolar couplings. The approach benefits from the high sensitivity associated with 1.3 mm samples, more abundant information including Hα resonances, and the narrow proton linewidths encountered for highly deuterated proteins. The labeling strategy solves amide proton exchange problems commonly encountered for membrane proteins when using perdeuteration and backexchange protocols, allowing access to alpha and all amide protons including those in exchange-protected regions. The incorporation of Hα protons provides new insights, as the close Hα–Hα and Hα–HN contacts present in β-sheets become accessible, improving the chance to determine the protein structure as compared with HN–HN contacts alone. Protonation of the Hα position higher than 90% is achieved for Ile, Leu, Phe, Tyr, Met, Val, Ala, Gln, Asn, Thr, Ser, Glu, Asp even though LAAO is only active at this degree for Ile, Leu, Phe, Tyr, Trp, Met. Additionally, the glycine methylene carbon is labeled preferentially with a single deuteron, allowing stereospecific assignment of glycine alpha protons. In solution, we show that the high deuteration level dramatically reduces R2 relaxation rates, which is beneficial for the study of large proteins and protein dynamics. We demonstrate the method using two model systems, as well as a 32 kDa membrane protein, hVDAC1, showing the applicability of the method to study membrane proteins. [ABSTRACT FROM AUTHOR]

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 RT 2 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 H 2 O 2 , and is also capable of modulating genes related to the DNA repair system and antioxidant pathways. [ABSTRACT FROM AUTHOR]

Abstract: L‐amino acid oxidase (LAAO) is able to stereoselectively catalyse the deamination of L‐amino acids to α‐keto acids, ammonia and H2O2. It has been reported that this enzyme has biological functions including inhibition of bacteria and antiviral, anti‐parasitic and anti‐tumour activities. To date, marine animal LAAO are constantly being identified. L‐amino acid oxidase derived from marine animals has been purified for Sebastes schlegelii, Platichthys stellatus, Gadus morhua, Signanus oramin, Aplysia californica and some others. In this paper, we review the reported progress of research on marine animal LAAO, including basic characteristics, tissue distribution and biological function. We intended to provide a reference for the further study of LAAO in marine animals. [ABSTRACT FROM AUTHOR]

L-amino acid oxidases (LAAOs) are ubiquitous enzymes in nature. Bioactivities described for these enzymes include apoptosis induction, edema formation, induction or inhibition of platelet aggregation, as well as antiviral, antiparasite, and antibacterial actions. With over 80 species, Micrurus snakes are the representatives of the Elapidae family in the New World. Although LAAOs in Micrurus venoms have been predicted by venom gland transcriptomic studies and detected in proteomic studies, no enzymes of this kind have been previously purified from their venoms. Earlier proteomic studies revealed that the venom of M. mipartitus from Colombia contains ~4% of LAAO. This enzyme, here named MipLAAO, was isolated and biochemically and functionally characterized. The enzyme is found in monomeric form, with an isotope-averaged molecular mass of 59,100.6 Da, as determined by MALDI-TOF. Its oxidase activity shows substrate preference for hydrophobic amino acids, being optimal at pH 8.0. By nucleotide sequencing of venom gland cDNA of mRNA transcripts obtained from a single snake, six isoforms of MipLAAO with minor variations among them were retrieved. The deduced sequences present a mature chain of 483 amino acids, with a predicted pI of 8.9, and theoretical masses between 55,010.9 and 55,121.0 Da. The difference with experimentally observed mass is likely due to glycosylation, in agreement with the finding of three putative N-glycosylation sites in its amino acid sequence. A phylogenetic analysis of MmipLAAO placed this new enzyme within the clade of homologous proteins from elapid snakes, characterized by the conserved Serine at position 223, in contrast to LAAOs from viperids. MmipLAAO showed a potent bactericidal effect on S. aureus (MIC: 2 µg/mL), but not on E. coli. The former activity could be of interest to future studies assessing its potential as antimicrobial agent. [ABSTRACT FROM AUTHOR]

Colorectal carcinoma is one of the most common cancers in adults. As chemotherapy, the first-choice treatment for colorectal carcinoma, is often infeasible due to acquired tumor resistance and several adverse effects, it is important to discover and explore new molecules with better therapeutic action. Snake venom toxins have shown promising results with high cytotoxicity against tumor cells, but their mechanisms of action remain unclear. Here we examined how BjussuLAAO-II, an L-amino acid oxidase isolated from Bothrops jararacussu snake venom, exerts cytotoxicity towards colorectal adenocarcinoma human cells (Caco-2) and human umbilical vein endothelial cell line (HUVEC). A 24-h treatment with BjussuLAAO-II at 0.25 − 5.00 μg/mL diminished cell viability by decreasing (i) mitochondrial activity, assessed by reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and resazurin; (ii) the activity of acid phosphatases; and (iii) lysosomal function, assessed by neutral red uptake. BjussuLAAO-II also increased intracellular levels of reactive oxygen species and DNA damage, as assessed by fluorescence and the comet assay, respectively. BjussuLAAO-II altered the expression of cell proliferation-related genes, as determined by RT-qPCR: it elevated the expression of the inflammatory cytokine genes TNF and IL6 , and lowered the expression of the apoptotic-related genes BAX , BCL2 , and RELA . Therefore, BjussuLAAO-II induces Caco-2 cells death by acting on multiple intracellular targets, providing important data for further studies to assess whether these effects are seen in both tumor and normal cells, with the aim of selecting this drug for possible therapeutic purposes in the future. [ABSTRACT FROM AUTHOR]

Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes α-imino acids generated by L- or D-amino acid oxidases with a preference for those deriving from Ala > Leu = L-Met > L-Gln, whereas it was poorly active on L-Phe and L-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 °C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms. [ABSTRACT FROM AUTHOR]