Cytotoxic L-amino acid oxidase from Bothrops moojeni: biochemical and functional characterization

An L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) was purified to a high degree using sequential CM-Sepharose ion-exchange and phenyl-Sepharose chromatography. When analyzed by mass spectrometry, the purified BmooLAAO-I presented a molecular weight of 64,889 and 130,779 under denaturing and nondenaturing conditions, respectively. BmooLAAO-I is a homodimeric acidic glycoprotein with a pI approximately 4.7, and the N-terminal sequence shows close structural similarity to other snake venom LAAOs. This enzyme was inactivated by freezing or low pH, and secondary structural analysis by circular dichroism revealed 48% alpha-helix, 20% beta-sheet, 12% beta-turn, and 20% random coil structures. BmooLAAO-I exhibited bactericidal, antitumoral, trypanocidal, edematogenic, and platelet-aggregating activities. All of these effects were inhibited by catalase, suggesting that these biological effects are mediated by the production of H(2)O(2). BmooLAAO-I induced typical apoptotic DNA fragmentation in HL-60 cells, which was also inhibited by catalase. These results point to the potential use of BmooLAAO-I as a therapeutic agent for treatment of diseases in which induction of H(2)O(2) production can be beneficial.     

蛇毒L-氨基酸氧化酶酶学性质及对血小板功能的影响

L-氨基酸氧化酶(EC1·4·3·2)是一种黄素类蛋白酶,其酶活性为立体特异性催化L-氨基酸的氧化脱氨,生成α-酮酸,同时产生氨和过氧化氢。酶广泛存在于多种生物体种类中。蛇毒L-氨基酸氧化酶(LAAO)是该类酶中研究最为深入的一族,因其易于纯化而成为研究L-氨基酸氧化酶的酶学、结构生物学和药物学的有趣客体。近年来,越来越多的具有不同分子量、不同底物选择性的蛇毒LAAO被分离纯化,国内外文献越来越多的报道了蛇毒LAAO的特性,如准确的分子量、底物特性、与血小板的相互作用、诱发出血和凋亡。本文就蛇毒LAAO的性质及其对血小板功能的…     

Biochemical and functional characterization of an L-amino acid oxidase isolated from Bothrops pirajai snake venom

In this work we describe the isolation of a new l-amino acid oxidase (LAAO) referred to as BpirLAAO-I from Bothrops pirajai snake venom, which was highly purified using a combination of molecular exclusion, affinity, and hydrophobic chromatography steps. BpirLAAO-I homodimeric acid glycoprotein (approximate Mr and pI of 130,000 and 4.9, respectively) displays high specificity toward hydrophobic/aromatic amino acids, while deglycosylation does not alter its enzymatic activity. The N-terminal LAAO sequence of its first 49 amino acids presented a high similarity between a amino acid sequence with other LAAOs from: Bothrops spp., Crotalus spp., Calloselasma rhodostoma, Agkistrodon spp., Trimeresurus spp., Pseudechis australis, Oxyuranus scutellatus, and Notechis scutatus. BpirLAAO-I induces time-dependent platelet aggregation, mouse paw edema, cytotoxic activity against Escherichia coli, Pseudomonas aeruginosa, Leishmania sp., and tumor cells, and also a typical fago (M13mp18) DNA fragmentation. Platelet aggregation, leishmanicidal and antitumoral activities were reduced by catalase. Thus, BpirLAAO-I is a multifunctional protein with promising biotechnological and medical applications.     

Enzymatic reaction of beta-N-methylaminoalanine with L-amino acid oxidase

The reaction of beta-N-methylaminoalanine (BMAA) with L-amino acid oxidase (L-AAO) in the presence of catalase yields ammonia and beta-N-methylaminopyruvate, which was trapped as its 2,4-dinitrophenylhydrazone, as products. Incubation of BMAA with L-AAO in the presence of semicarbazide led to the formation of a semicarbazone, indicating intermediate iminium ion formation; when potassium cyanide (5 mM) was added, semicarbazone formation was blocked. The formation of beta-N-methylaminopyruvate was decreased by omission of catalase and was reduced in the presence of hydrogen peroxide (100 mM). These results indicate that BMAA is converted by L-AAO to the corresponding alpha-imino acid, which undergoes hydrolysis to beta-N-methylaminopyruvate. The alpha-keto acid is readily oxidized to N-methylglycine by hydrogen peroxide.     

Purification and characterization of L-amino acid oxidase from the solid-state grown cultures of Aspergillus oryzae ASH

L-Amino acid oxidase (L-AAO) was purified from the solid state-grown cultures of A. oryzae ASH (JX006239.1) by fractional salting out, followed by ion exchange and gel filtration chromatography, to its molecular homogeneity, displaying 3.38-fold purification in comparison with the crude enzyme. SDS-PAGE revealed the enzyme to be a homo-dimer with ～55-kDa subunits, with approximate molecular weight on native PAGE of 105–110 kDa. Two absorption maxima, at 280 nm and 341 nm, for the apoproteinic and FMN prosthetic group of the enzyme, respectively, were observed, with no detected surface glycosyl residues. The enzyme had maximum activity at pH 7.8–8.0, with ionic structural stability within pH range 7.2–7.6 and pH precipitation point (pI) 4.1–5.0. L-AAO exhibited the highest activity at 55°C, with plausible thermal stability below 40°C. The enzyme had T _1/2 values of 21.2, 8.3, 3.6, 3.1, 2.6 h at 30, 35, 40, 50, 60°C with Tm 61.3°C. Kinetically, A. oryzae L-AAO displayed a broad oxidative activity for tested amino acids as substrates. However, the enzyme had a higher affinity towards basic amino acid L-lysine (K _m 3.3 mM, K _cat 0.04 s^?1) followed by aromatic amino acids L-tyrosine (K _m 5.3 mM, K _cat 0.036 s^?1) and L-phenylalanine (K _m 6.6 mM), with 1ow affinity for the S-amino acid L-methionine (K _m 15.6 mM). The higher specificity of A. oryzae L-AAO to L-lysine as substrate seems to be a unique property comparing to this enzyme from other microbes. The enzyme was significantly inhibited by hydroxylamine and SDS, with slight inhibition by EDTA. The enzyme had a little effect on AST and ALT, with no effect on platelet aggregation and blood hemolysis in vivo with an obvious cytotoxic effect towards HepG2 (IC₅₀ 832.2 μg/mL) and MCF-7 (IC₅₀, 370.6 μg/mL) tumor cells in vitro.     

Characterization and expression of L-amino acid oxidase of mouse milk

l-Amino acid oxidase (LAO) was purified from mouse milk. LAO reacted with l-amino acids in an apparent order of Phe > Met, Tyr > Cys, Leu > His other 11 amino acids tested and produced H(2)O(2) in a dose- and time-dependent manner. LAO in milk had a molecular mass of about 113 kDa and was converted to a 60-kDa protein by SDS-PAGE. LAO consisted of two subunits. The N- and C-terminal amino acid sequence determination followed by cDNA cloning showed that the 60-kDa protein consisted of 497 amino acids. LAO mRNA spanned about 2.0 kb, and its expression was found only in the mammary epithelial cells. Glucocorticoid was essential for LAO gene expression. Thus, the LAO gene is expressed acutely upon the onset of milk synthesis. LAO mRNA increased 1 day before parturition, peaked during early to mid-lactation, and decreased at the end of lactation. This is the first demonstration showing that LAO is present in milk. Mastitis is caused by an intramammary bacterial infection. As mouse milk produced H(2)O(2) using endogenous free amino acids, we suggest that LAO, together with free amino acids, is responsible for killing bacteria in the mammary gland.     

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.     

Purification and characterization of epsilon-N-trimethyllysine L-amino oxidase from Neurospora crassa.

epsilon-N-Trimethyllysine L-amino oxidase from Neurospora crassa has been purified to electrophoretic homogeneity. A 1500-fold purification was obtained by centrifugation and successive column chromatography on ion-exchange and gel filtration supports. The enzyme has an estimated molecular weight of 160 000. It transforms epsilon-N-trimethyllysine into alpha-keto, epsilon-N-trimethylhexanoic acid by oxidative deamination. Kinetic studies of this new enzyme are reported and its probable physiological role is discussed.