Substrate specificity of king cobra (Ophiophagus hannah) venom L-amino acid oxidase

1. Substrate specificity of purified king cobra (Ophiophagus hannah) venom L-amino acid oxidase was investigated. 2. The enzyme was highly specific for the L-enantiomer of amino acid. Effective oxidation of L-amino acid by the enzyme requires the presence of a free primary alpha-amino group but the alpha-carboxylate group is not as critical for the catalysis. 3. The enzyme was very active against L-Lys, L-Phe, L-Leu, L-Tyr, L-Tryp, L-Arg, L-Met, L-ornithine, L-norleucine and L-norvaline and moderately active against L-His, L-cystine and L-Ileu. Other L-amino acids were oxidized slowly or not oxidized. 4. The data suggest the presence of a side chain binding site in the enzyme, and that the binding site comprises at least five 'subsites': the hydrophobic subsites a, b and c; and the two 'amino' binding subsites d and e. Subsite b appears to be able to accommodate two methylene/methyl carbons.     

Isolation and characterization of an apoptotic and platelet aggregation inhibiting L-amino acid oxidase from Vipera berus berus (common viper) venom

An L-amino acid oxidase was isolated from the venom of the common viper Vipera berus berus by a three-step procedure combining gel filtration, ion exchange and hydrophobic chromatography. The enzyme is a non-covalently bound homodimer with a monomeric molecular mass of 57.7 kDa. The N-terminal amino acid sequence and the internal peptide sequences show close structural homology with other snake venom L-amino acid oxidases. The purified protein catalyzed oxidative desamination of L-amino acids, the most specific substrate is L-Phe. The best substrates among the studied 20 amino acids were: L-Met, L-Leu, L-Phe, L-Ile, L-Arg and L-His. Five amino acids, L-Ser, L-Pro, Gly, L-Thr and L-Cys, were not oxidized. The enzyme inhibited ADP-induced platelet aggregation dose-dependently with an IC50 of 0.07 microM. The effect was neutralized by catalase. V. berus berus LAAO induced apoptosis in cultured HeLa and K562 cells as shown by DNA fragmentation gel pattern. The induction of apoptosis was inhibited by catalase.     

Isolation and structural characterization of a cytotoxic L-amino acid oxidase from Agkistrodon contortrix laticinctus snake venom: preliminary crystallographic data.

We have purified a cytotoxic L-amino acid oxidase (LAO) from Agkistrodon contortrix laticinctus snake venom by means of Superdex-200 gel filtration, followed by phenyl-Sepharose CL-4B chromatography. The purified enzyme (ACL LAO) is a dimer on gel filtration, with a M(r) of 60,000 for the monomer as estimated by SDS-PAGE. LAO activity was tested against 15 amino acids, but only 9 were oxidized by the enzyme, suggesting that it presents some degree of specificity. ACL LAO has apoptosis-inducing activity in an HL-60 cell culture assay. After 24 h treatment with 25 micrograms/ml of ACL LAO, the typical DNA fragmentation pattern of apoptotic cells was observed on agarose gel electrophoresis. NMR analysis showed the presence of a flavin mononucleotide prosthetic group. To solve its 3-D structure, crystals of the purified protein were grown in 0.1 M Tris-HCl, pH 8.5, and 2 M (NH(4))(2)SO(4). Diffraction data collected to 3.5 A showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 A, c = 183.45 A. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase.     

Isolation and characterization of an unusual form of L-amino acid oxidase from King cobra (Ophiophagus hannah) venom

The L-amino acid oxidase (EC 1. 4. 3. 2) from King cobra (Ophiophagus hannah) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was determined to be 140000 when examined by gel filtration and 68000 by SDS-polyacrylamide gel electrophoresis. The enzyme had an isoelectric point of 4.5 and an intravenous LD50 of 5 micrograms/g in mice. It is a glycoprotein and contains two moles of FAD per mole of enzyme. The enzyme exhibited unusual thermal stability and unlike most other venom L-amino acid oxidases, it was stable in alkaline solution and was not inactivated by freezing.     

江浙蝮蛇（Agkistrodon halys Pallas）毒的抑菌作用及其抑菌组 …

This paper reports the venom from Agkistrodon halys Pallas have inhibitory activity against fungi and E. coli by tested in a disc diffusion assay. An antibacterial component--LAO from the venom were purified to homogeneous. It had not only antibacterial effect, but L-amino acid oxidase activity. And its enzymatic specific activity was 808 U/mg. The venom had at least 3 antibacterial components (I, II, III) as determined by acid polyacrilamide gel electrophoresis, LAO is the antibaterial components II.     

Isolation, structural, and functional characterization of an apoptosis-inducing L-amino acid oxidase from leaf-nosed viper (Eristocophis macmahoni) snake venom

The enzyme L-amino acid oxidase (LAO) from the leaf-nosed viper (Eristocophis macmahoni) snake venom was purified to homogeneity in a single step using high performance liquid chromatography on a Nucleosil 7C18 reverse phase column. The molecular mass of the purified enzyme was 58734.0 Da, as determined by matrix-assisted laser desorption/ionization mass spectrometry. The N-terminal amino acid sequence (ADDKNPLEEAFREADYEVFLEIAKNGL) and the chemical composition of the purified LNV-LAO shows close structural homology with other L-amino acid oxidases isolated from different snake venoms. The secondary structural contents analysis of LAO, established by means of circular dichroism, revealed ca. 49% alpha-helix, 19% beta-sheet, 10% beta-turn, and 22% random coil structure. The purified LNV-LAO not only retained its specific enzymatic activity (73.46 U/mg), determined against L-leucine as a substrate, but also exhibited potent haemolytic (1-10 microg/ml), edema- (MED 4.8 microg/ml) and human platelet aggregation-inducing (ED50 33 microg/ml) properties. Unlike other haemorrhagic snake venom L-amino acid oxidases, the LNV-LAO does not produce haemorrhage. In addition to these local effects, the purified LNV-LAO showed apoptosis-inducing activity in the MM6 cell culture assay. After 18 h treatment with 25-100 microg/ml of LAO, the typical DNA fragmentation pattern of apoptotic cells was observed by means of fluorescent microscopy and agarose gel electrophoresis.     

Vinylglycine and proparglyglycine: complementary suicide substrates for L-amino acid oxidase and D-amino acid oxidase.

Proparglyglycine (2-amino-4-pentynoate) and vinylglycine (2-amino-3-butenoate) have been examined as substrates and possible inactivators of two flavo enzymes, D-amino acid oxidase from pig kidney and L-amino acid oxidase from Crotalus adamanteus venom. Vinylglycine is rapidly oxidized by both enzymes but only L-amino acid oxidase is inactivated under assay conditions. The loss of activity probably involves covalent modification of an active site residue rather than the flavin adenine dinucleotide coenzyme and occurs once every 20000 turnovers. We have confirmed the recent observation (Horiike, K, Hishina, Y., Miyake, Y., and Yamano, T. (1975) J, Biochem. (Tokyo), 78, 57) that D-proparglglycine is oxidized with a time-dependent loss of activity by D-amino acid oxidase and have examined some mechanistic aspects of this inactivation, The extent of residual oxidase activity, insensitive to further inactivation, is about 2%, at which point 1.7 labels/subunit have been introduced with propargly[2-14C]glycine as substrate. L-Proparglyclycine is a substrate but not an inactivator of L-amino acid oxidase and the product ahat accumulats in the nonnucleophilic N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer is acetopyruvate. In the presence of butylamine HCl, a species with lambdaman 317 nm (epsilon = 15 000) accumulates that may be a conjugated eneamine adduct. The same species accumulates from D-amino acid oxidase oxidation of D-propargylglycine prior to inactivation; the inactivated apo D-amino acid oxidase has a new peak at 317 nm that is probably a similar eneamine. A likely inactivating species is 2-keto-3,4-pentadienoate arising from facile rearrangement of the expected initial product 2-keto 4 pentynoate. Vinylglycine and proparglyglycine show inactivation specificity, then, for L-and D-amino acid oxidase, respectively.     

Biochemical composition, lethality and pathophysiology of venom from two cobras-- Naja naja and N. kaouthia

The cobras Naja naja and N. kaouthia are abundant in eastern and north-eastern India, accounting for maximum snakebite deaths. Here we report on variation in the composition of Naja kaouthia and N. naja venom from eastern India on corresponding differences in the severity of pathogenesis. These two venoms differ in chromatographic elution profile through Sephadex G-50 and enzyme activity, protein and carbohydrate contents associated with each fraction. The presence of greater amounts of basic phospholipase A2, L-amino acid oxidase and low molecular weight membrane active polypeptides in the N. naja venom makes it more toxic than N. kaouthia venom. A commercial polyvalent antivenom raised against N. naja venom inactivates lethality and variety of toxic effects of homologous venom more effectively than N. kaouthia venom.     

Preparation and characterization of isolubilized L-amino acid oxidase

The enzyme L-amino acid oxidase of Crotalus adamanteus was covalently coupled to porous 96% silica glass particles. The insolubilized enzyme was active on several L-amino acids including: leucine, isoleucine, cysteine, phenylalanine, tryptophane, and methionine. No activity was observed with D-amino acids, L-asparagine, or L-proline. Maximum activity was observed at pH 7.8. Stability of the enzyme derivative was demonstrated by continuous operation of an enzyme column for 35 days, during which the bound enzyme oxidized over 5000 times its own weight of substrate.     

Rat kidney L-alpha-hydroxy acid oxidase: isolation of enzyme with one flavine coenzyme per two subunits.

L-alpha-Hydroxy acid oxidase (listed as EC 1.4.3.2, L-amino acid: O2 oxidoreductase) has been purified 100-fold from rat kidney to apparent homogeneity by gel electrophoresis. A subunit molecular weight of 47,500 was found by sodium dodecyl sulfate gel electrophoresis, but in contrast to previous reports, the enzyme has been found to have a molecular weight of ca. 200,000 by Sephadex gel filtration and by dodecyl sulfate gel electrophoresis of the enzyme cross-linked with dimethyl suberimidate. A somewhat higher value was found by sedimentation equilibrium, but a tetrameric structure for the active enzyme is definitely established. The enzyme was found to contain the FMN coenzyme at a concentration of one FMN/102,000 daltons or one flavine/two subunits, a highly unusual finding. This ratio was determined from spectroscopic analysis of the FMN in lyophilized samples of the enzyme and by titration of the coenzyme with the flavine specific enzyme inactivator 2-hydroxy-3-butynoate. The enzyme has the same specific activity as a crystalline sample of the enzyme reported to have twice as much flavine/milligram.     

Two L-amino acid oxidase isoenzymes from Russell's viper (Daboia russelli russelli) venom with different mechanisms of inhibition by substrate analogs

Two isoforms, L(1) and L(2), of L-amino acid oxidase have been isolated from Russell's viper venom by Sephadex G-100 gel filtration followed by CM-Sephadex C-50 ion exchange chromatography. The enzymes, with different isoelectric points, are monomers of 60-63 kDa as observed from size exclusion HPLC and SDS/PAGE. Partial N-terminal amino acid sequencing of L(1) and L(2) showed significant homology with other snake venom L-amino acid oxidases. Both the enzymes exhibit marked substrate preference for hydrophobic amino acids, maximum catalytic efficiency being observed with L-Phe. Inhibition of L(1) and L(2) by the substrate analogs N-acetyltryptophan and N-acetyl-L-tryptophan amide has been followed. The initial uncompetitive inhibition of L(1) followed by mixed inhibition at higher concentrations suggested the existence of two different inhibitor-binding sites distinct from the substrate-binding site. In the case of L(2), initial linear competitive inhibition followed by mixed inhibition suggested the existence of two nonoverlapping inhibitor-binding sites, one of which is the substrate-binding site. An inhibition kinetic study with O-aminobenzoic acid, a mimicking substrate with amino, carboxylate and hydrophobic parts, indicated the presence of three and two binding sites in L(1) and L(2), respectively, including one at the substrate-binding site. An inhibitor cross-competition kinetic study indicated mutually excluding binding between N-acetyltryptophan, N-acetyl-L-tryptophan amide and O-aminobenzoic acid in both the isoforms, except at the substrate-binding site of L(1). Binding of substrate analogs with different electrostatic and hydrophobic properties provides useful insights into the environment of the catalytic sites. Furthermore, it predicts the minimum structural requirement for a ligand to enter and anchor at the respective functional sites of LAAO that may facilitate the design of suicidal inhibitors.     

Mechanisms of inactivation of molybdoenzymes by cyanide

The reduced forms of xanthine oxidase, xanthine dehydrogenase, aldehyde oxidase, and sulfite oxidase are inactivated by cyanide. Following gel filtration to remove excess of reductant and cyanide, the isolated enzymes remain inactive. Thiocyanate, a product of inactivation of the oxidized forms of the xanthine- and aldehyde-oxidizing enzymes by cyanide, is not released during inactivation of the reduced enzymes. Studies with [14C]cyanide show that, while stoichiometric binding is required for the onset of inactivation, its continued binding is not essential to maintenance of the inactivated state. Electron paramagnetic resonance and absorption spectroscopic studies on the isolated inactivated enzymes show that prosthetic groups other than molybdenum are fully oxidized but that the molybdenum centers are modified. Reactivation is accomplished by incubation with suitable oxidants. Aerobic reactivation of inactive sulfite oxidase required only 1 eq of ferricyanide/active site. However, under rigorously anaerobic conditions, 3 to 4 mol of ferricyanide/active site were reduced, indicating that the molybdenum centers in the inactive enzyme had been reduced below the levels attained by the native enzyme during catalysis.     

Oxidative deamination of S-aminopropylcysteine and S-aminoethylhomocysteine.

S-aminopropylcysteine and S-aminoethylhomocysteine are oxidized by snake venom L-amino acid oxidase in the presence of catalase with formation of the respective ketoderivatives. Only the ketoderivative of S-aminopropylcysteine cyclizes to give a seven membered ring (ketimine) absorbing at 296 nm. In the absence of catalase both ketoderivatives are oxidatively decarboxylated.     

SO-LAAO, a novel L-amino acid oxidase that enables Streptococcus oligofermentans to outcompete Streptococcus mutans by generating H2O2 from peptone

We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans, the primary cariogenic pathogen, by producing hydrogen peroxide (H(2)O(2)) through lactate oxidase activity. In this study, we found that the lox mutant of S. oligofermentans regained the inhibition while growing on peptone-rich plates. Further studies demonstrated that the H(2)O(2) produced on peptone by S. oligofermentans was mainly derived from seven L-amino acids, i.e., L-aspartic acid, L-tryptophan, L-lysine, L-isoleucine, L-arginine, L-asparagine, and L-glutamine, indicating the possible existence of L-amino acid oxidase (LAAO) that can produce H(2)O(2) from L-amino acids. Through searching the S. oligofermentans genome for open reading frames with a conserved flavin adenine dinucleotide binding motif that exists in the known LAAOs, including those of snake venom, fungi, and bacteria, a putative LAAO gene, assigned as aao(So), was cloned and overexpressed in Escherichia coli. The purified protein, SO-LAAO, showed a molecular mass of 43 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and catalyzed H(2)O(2) formation from the seven L-amino acids determined above, thus confirming its LAAO activity. The SO-LAAO identified in S. oligofermentans differed evidently from the known LAAOs in both substrate profile and sequence, suggesting that it could represent a novel LAAO. An aao(So) mutant of S. oligofermentans did lose H(2)O(2) formation from the seven L-amino acids, further verifying its function as an LAAO. Furthermore, the inhibition by S. oligofermentans of S. mutans in a peptone-rich mixed-species biofilm was greatly reduced for the aao(So) mutant, indicating the gene's importance in interspecies competition.     

Molecular cloning and functional analysis of apoxin I, a snake venom-derived apoptosis-inducing factor with L-amino acid oxidase activity

We previously purified apoxin I, an apoptosis-inducing factor with L-amino acid oxidase (LAO) activity, from Western diamondback rattlesnake venom. To determine the primary structure of apoxin I, we cloned its cDNA. The amino acid sequence showed that apoxin I has an FAD binding domain and shares homology with L-amino acid oxidase (LAO) from Neurospora crassa, human monoamine oxidase B, and mouse interleukin 4-induced F1G1 protein. The full-length apoxin I has an N-terminal signal sequence that is processed in mature apoxin I in venom. When the apoxin I gene was transfected into human 293T cells, the recombinant protein was expressed in the cells, and a significant amount of apoxin I was secreted into the medium. The secreted recombinant apoxin I protein showed LAO and apoptosis-inducing activity, but the recombinant protein in the cells did not, suggesting that maturation and secretion of the apoxin I protein is needed for its activity. Treating the transfected cells with tunicamycin inhibited the secretion and LAO activity of the recombinant apoxin I. In addition, deleting the amino-terminal region flanking the signal sequence, the FAD-binding domain and the carboxy-terminal region abolished the secretion and LAO activity of the recombinant proteins. These results indicate that in order for apoxin I to become active, these regions and posttranslational modification, such as N-glycosylation, are required.     

Localization of some enzymes in the main venom glands of viperid snakes

Several secretory and nonsecretory enzymes were localized histochemically in the main venom gland of 13 viperid snakes. All secretory cells show the intracellular oxidative enzymes succinate dehydrogenase and monoamine oxidase. The granular reactions obtained for both enzymes resemble mitochondria in distribution. Distinctive cells with a very high succinate dehydrogenase activity are dispersed among the secretory cells of all species except Atractaspis. Nonspecific acid phosphatase activity is found in the supranuclear region of the secretory cells in species that do not secrete this enzyme and throughout the cytoplasm in snakes that secrete the enzyme. Nonspecific alkaline phosphatase activity occurs in the secretory cells of those snakes whose venom shows this activity. Leucine amino peptidase (aryl amidase) activity is found in the venom and in the secretory cells of all the species. In Vipera palaestinae both the venom and the secretory cells of the main venom gland contain nonspecific esterase, L-amino acid oxidase and phosphodiesterase activities. The localization of phosphodiesterase and L-amino acid oxidase do not show major differences between glands at different intervals from an initial milking. Adenosine-monophosphate phosphatase activity is localized in the supranuclear region of the secretory cells in the glands of Vipera palaestinae and Aspis cerastes. Its activity is found in the venom of Aspis only.     

Studies on the reaction of D-amino acid oxidase with beta-cyano-D-alanine. Observation of an intermediary stable charge transfer complex

The reaction of D-amino acid oxidase [EC 1.4.3.3] (DAO) from porcine kidney with beta-cyano-D-alanine (D-BCNA) was studied. DAO was found to catalyze elimination of the cyano group as well as oxidation of D-BCNA. During the course of the reaction in the presence of excess oxygen, an intermediate was observed which exhibited a characteristic absorption spectrum with a broad charge transfer band in the longer wavelength region. The CD spectrum of this intermediate resembles that of DAO-anthranilate complex. The rate of oxygen consumption in the aerobic reaction decreased with time, suggesting product inhibition due to complex formation between the enzyme and the product. Anaerobic addition of D-BCNA reduced the enzyme to its fully reduced state, the CD spectrum of which closely resembles that of the enzyme reduced by excess D-alanine. When an appropriate amount of D-BCNA was added to the enzyme under air, the charge transfer complex was observed immediately, and underwent a change to the reduced state as the oxygen was consumed. The binding strength in the charge transfer complex was found to be comparable to that in DAO-benzoate complex. The accumulating product in the oxidation of D-BCNA had a strong absorption at 285 nm. The aerobic reaction of beta-cyano-L-alanine (L-BCNA) with snake venom L-amino acid oxidase (LAO) produced the same product with an absorption at 285 nm as the reaction of DAO with D-BCNA. The product obtained in the reaction with LAO was found to form the same charge transfer complex with DAO. We tentatively identified this product as alpha-amino-beta-cyanoacrylate and the charge transfer complex as the complex of alpha-amino-alpha-cyanoacrylate with the oxidized enzyme. A hypothetical reaction pathway based on the present finding is proposed. Addition of L-BCNA to the enzyme produced an absorption spectrum very similar to that of the DAO-benzoate complex without oxidation or elimination. L-BCNA was found to be a competitive inhibitor of the oxidation of D-alanine.     

Cyanide formation from histidine in Chlorella. A general reaction of aromatic amino acids catalyzed by amino acid oxidase systems.

The formation of HCN from D-histidine in Chlorella vulgaris extracts is shown to be due to the combined action of a soluble protein and a particulate component. Either horse-radish peroxidase (EC 1.11.1.7) or a metal ion with redox properties can be substituted for the particulate component. Ions of manganese and vanadium are especially effective, as are o-phenanthroline complexes of iron. Cobalt ions are less active. The D-amino acid oxidase (EC 1.4.3.3) from kidney and the L-amino acid oxidase (EC 1.4.3.2) from snake venom likewise cause HCN production from histidine when supplemented with the particulate preparation from Chlorella or with peroxidase or with a redox metal ion. The stereospecificity of the amino acid oxidase determines which of the two stereoisomers of histidine is active as an HCN precursor. Though histidine is the best substrate for HCN production, other naturally occurring aromatic amino acids (viz. tyrosine, phenylalanine and tryptophan) can also serve as HCN precursors with these enzyme systems. The relative effectiveness of each substrate varies with the amino acid oxidase enzyme and with the supplement. With respect to this latter property, the particulate preparation from Chlorella behaves more like a metal ion than like peroxidase.     

烙铁头蛇毒L—氨基酸氧化酶的分离、纯化及其生物学活性

通过DEAESephadexA 5 0阴离子交换柱 ,SephadexG 75分子筛 ,ResourseQ阴离子交换柱三步层析从湖南产的烙铁头蛇毒中分离、纯化得到一个L 氨基酸氧化酶 (TM LAO) ,它由两个非共价的亚基组成 ,每个亚基的分子量为 5 5kD。与台湾产的烙铁头蛇毒L 氨基酸氧化酶分子量 ( 70kD)不同。TM LAO的N末端氨基酸序列是ADNKNPLEECFRETNYEEFLEIAR ,与报道的蝰科的L 氨基酸氧化酶的相似性比眼镜蛇科的要高。TM LAO能抑制大肠杆菌、金黄色葡萄球菌和痢疾杆菌的生长 ,杀死肿瘤细胞以及诱导血小板聚集。这些活性能被过氧化氢酶所抑制 ,说明TM LAO生理学功能主要是通过酶反应产生的过氧化氢 (H2 O2 )介导的