In-gel detection of L-amino acid oxidases based on the visualisation of hydrogen peroxide production

A simple and practicable method for in-gel detection of bacterial produced L-amino acid oxidases (L-AAOs) after non-denaturing SDS-PAGE based on the visualisation of occurring hydrogen peroxide production is described. Advantages of this screening method for L-AAOs are the determination of their numbers and approximate molecular weights additionally in one approach.     

Hydrogen peroxide produced by two amino acid oxidases mediates antibacterial actions

The antibacterial actions of two amino acid oxidases, a D-amino acid oxidase from hog kidney and a L-amino acid oxidase from the venom of Agkistrodon halys, were investigated, demonstrating that both enzymes were able to inhibit the growth of both Gram-positive and Gram-negative bacteria, and that hydrogen peroxide, a product of their enzymatic reactions, was the antibacterial factor. However, hydrogen peroxide generated in the enzymatic reactions was not sufficient to explain the degree to which bacterial growth was inhibited. A fluorescence labeling assay showed that both of these two enzymes could bind to the surfaces of bacteria. To the best of our knowledge, this is the first report regarding the antibacterial activity of the D-amino acid oxidases.     

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.     

A thermostable L-aspartate oxidase: a new tool for biotechnological applications

L-Amino acid oxidases (LAAOs) are homodimeric flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Unlike the D-selective counterpart, the biotechnological application of LAAOs has not been thoroughly advanced because of the difficulties in their expression as recombinant protein in prokaryotic hosts. In this work, L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, specific for L-aspartate and L-asparagine only) was efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. This recombinant flavoenzyme shows the classical properties of FAD-containing oxidases. Indeed, StLASPO shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80 °C) and high temperature optimum, stable activity in a broad range of pH (7.0–10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. This latter property significantly distinguishes StLASPO from the E. coli counterpart. StLASPO represents an appropriate novel biocatalyst for the production of D-aspartate and a well-suited protein scaffold to evolve a LAAO activity by protein engineering.     

l-Amino acid oxidases from microbial sources: types,properties, functions,and applications

l-Amino acid oxidases (LAAOs), which catalyze the stereospecific oxidative deamination of l-amino acids to α-keto acids and ammonia, are flavin adenine dinucleotide-containing homodimeric proteins. l-Amino acid oxidases are widely distributed in diverse organisms and have a range of properties. Because expressing LAAOs as recombinant proteins in heterologous hosts is difficult, their biotechnological applications have not been thoroughly advanced. LAAOs are thought to contribute to amino acid catabolism, enhance iron acquisition, display antimicrobial activity, and catalyze keto acid production, among other roles. Here, we review the types, properties, structures, biological functions, heterologous expression, and applications of LAAOs obtained from microbial sources. We expect this review to increase interest in LAAO studies.     

L-氨基酸氧化酶的研究进展

L-氨基酸氧化酶(L-amino acid oxidase, LAAO)能特异性催化L-氨基酸氧化脱氨,生成α-酮酸、氨和H₂O₂。该酶分布较广,其中蛇毒源LAAO是该类酶中研究最为深入的一类,近年来,越来越多的非蛇毒源LAAO被发现和报道,现对蛇毒源和非蛇毒源LAAO的研究进展进行了综述。现有研究表明,不同物种来源的LAAO,其底物选择性、等电点、稳定性等理化性质不尽相同;虽对其结构的研究还较少,但现有的研究表明蛇毒源和非蛇毒源LAAO的结构都含有FAD结合结构域、底物结构域和螺旋结构域;研究已发现不同来源的LAAO体外具有多种不同的生物学功能,而这些生物学功能多数是由于其产物H₂O₂作用的结果;对LAAO异源表达的研究较少且都不甚成功,可能是由于其需要进行翻译后修饰。     

L-Lysine alpha-oxidase: physicochemical and biological properties

This review summarizes data on the properties of L-lysine -oxidase, an enzyme that belongs to the group of oxidases of L-amino acids. This enzyme acts virtually only on L-lysine with a rather low K _m yielding -keto--aminocaproic acid. The decrease in the level of the essential amino acid L-lysine and the formation of hydrogen peroxide during the reaction possibly provide the basis for the unique properties of L-lysine -oxidase: cytotoxic, antitumor, antimetastatic, antiinvasive, antibacterial, and antiviral activities, as well as an immunomodulating effect. Native L-lysine -oxidase and its immobilized forms are promising tools for determination of concentration of L-lysine in various biological materials.     

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.     

广西眼镜蛇毒L-氨基酸氧化酶体内外抗血小板聚集实验研究

目的 研究广西眼镜蛇中提取的L-氨基酸氧化酶(L-amino acid oxidase)在人体外及家兔体内的抗血小板聚集作用.方法 用比浊法测定广西眼镜蛇毒L-氨基酸氧化酶对二磷酸腺苷(ADP)、胶原、凝血酶、花生四烯酸(AA)在人体外及家兔体内引起的血小板聚集率的影响.结果 实验中,能明显抑制二磷酸腺苷(ADP)、胶原、凝血酶、花生四烯酸(AA)引起的血小板聚集,并呈明显的正相关.结论 广西眼镜蛇毒L-氨基酸氧化酶在体内外均有较强的抗血小板聚集活性.     

The structure of L-amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site

The structure of L-amino acid oxidase (LAAO) from Calloselasma rhodostoma has been determined to 2.0 A resolution in the presence of two ligands: citrate and o-aminobenzoate (AB). The protomer consists of three domains: an FAD-binding domain, a substrate-binding domain and a helical domain. The interface between the substrate-binding and helical domains forms a 25 A long funnel, which provides access to the active site. Three AB molecules are visible within the funnel of the LAAO-AB complex; their orientations suggest the trajectory of the substrate to the active site. The innermost AB molecule makes hydrogen bond contacts with the active site residues, Arg90 and Gly464, and the aromatic portion of the ligand is situated in a hydrophobic pocket. These contacts are proposed to mimic those of the natural substrate. Comparison of LAAO with the structure of mammalian D-amino acid oxidase reveals significant differences in their modes of substrate entry. Furthermore, a mirror-symmetrical relationship between the two substrate-binding sites is observed which facilitates enantiomeric selectivity while preserving a common arrangement of the atoms involved in catalysis.     

Structural insights into selectivity and cofactor binding in snake venom L-amino acid oxidases

l-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate l-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH₃) and hydrogen peroxide (H₂O₂). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1 Å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62–71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode.     

IL-4-induced gene-1 is a leukocyte L-amino acid oxidase with an unusual acidic pH preference and lysosomal localization

IL-4-induced gene-1 (Il4i1 or Fig1) initially isolated as a gene of unknown function from mouse B lymphocytes, is limited in expression to primarily immune tissues and genetically maps to a region of susceptibility to autoimmune disease. The predicted Il4i1 protein (IL4I1) sequence is most similar to apoptosis-inducing protein and Apoxin I, both l-amino acid oxidases (LAAO; Enzyme Commission 1.4.3.2). We demonstrate that IL4I1 has unique LAAO properties. IL4I1 has preference for aromatic amino acid substrates, having highest specific activity with phenylalanine. In support of this selectivity, IL4I1 is inhibited by aromatic competitors (benzoic acid and para-aminobenzoic acid), but not by nonaromatic LAAO inhibitors. Il4i1 protein and enzyme activity is found in the insoluble fraction of transient transfections, implying an association with cell membrane and possibly intracellular organelles. Indeed, IL4I1 has the unique property of being most active at acidic pH (pH 4), suggesting it may reside preferentially in lysosomes. IL4I1 is N-linked glycosylated, a requirement for lysosomal localization. Confocal microscopy of cells expressing IL4I1 translationally fused to red fluorescent protein demonstrated that IL4I1 colocalized with GFP targeted to lysosomes and with acriflavine, a green fluorescent dye that is taken up into lysosomes. Thus, IL4I1 is a unique mammalian LAAO targeted to lysosomes, an important subcellular compartment involved in Ag processing.     

Molecular approaches for structural characterization of Bothrops L-amino acid oxidases with antiprotozoal activity: cDNA cloning, comparative sequence analysis, and molecular modeling

Two l-amino acid oxidases (LAAOs) were identified by random sequencing of cDNA libraries from the venom glands of Bothrops moojeni(BmooLAAO) and Bothrops jararacussu(Bjussu LAAO). Phylogenetic analysis involving other SV-LAAOs showed sequence identities within the range 83-87% being closely related to those from Agkistrodon and Trimeresurus. Molecular modeling experiments indicated the FAD-binding, substrate-binding, and helical domains of Bmoo and Bjussu LAAOs. The RMS deviations obtained by the superposition of those domains and that from Calloselasma rhodostoma LAAO crystal structure confirm the high degree of structural similarity between these enzymes. Purified BjussuLAAO-I and BmooLAAO-I exhibited antiprotozoal activities which were demonstrated to be hydrogen-peroxide mediated. This is the first report on the isolation and identification of cDNAs encoding LAAOs from Bothrops venom. The findings here reported contribute to the overall structural elucidation of SV-LAAOs and will advance the understanding on their mode of action.     

The effect of Aerococcus viridans--the basis of the new therapeutic-prophylactic preparation M-bacterin--on the biological properties of Staphylococcus aureus

Representatives of the normal microflora from genus Aerococcus, in particular strain Aerococcus viridans 167 isolated from breast milk are studied for their effect on biological properties of Staphylococcus aureus in vitro and in vivo. It is established that the number of viable cells of the staphylococcus cultivated in the presence of antagonists in the beef-extract agar decreases progressively with each following passage, the population dying after the seventh-eight passage. Electronograms fix deep changes in the cell ultrastructure. A degree of changes in biological properties depends on the duration of the antagonist action. The results obtained reveal one of the mechanisms of the antagonistic action of aerococci-antagonists producing hydrogen peroxide.     

l-Amino acid oxidase as biocatalyst: a dream too far?

l-Amino acid oxidase (LAAO) is a flavoenzyme containing non-covalently bound flavin adenine dinucleotide, which catalyzes the stereospecific oxidative deamination of l-amino acids to α-keto acids and also produces ammonia and hydrogen peroxide via an imino acid intermediate. LAAOs purified from snake venoms are the best-studied members of this family of enzymes, although a number of LAAOs from bacterial and fungal sources have been also reported. From a biochemical point of view, LAAOs from different sources are distinguished by molecular mass, substrate specificity, post-translational modifications and regulation. In analogy to the well-known biotechnological applications of d-amino acid oxidase, important results are expected from the availability of suitable LAAOs; however, these expectations have not been fulfilled yet because none of the “true” LAAOs has successfully been expressed as a recombinant protein in prokaryotic hosts, such as Escherichia coli. In enzyme biotechnology, recombinant production of a protein is mandatory both for the production of large amounts of the catalyst and to improve its biochemical properties by protein engineering. As an alternative, flavoenzymes active on specific l-amino acids have been identified, e.g., l-aspartate oxidase, l-lysine oxidase, l-phenylalanine oxidase, etc. According to presently available information, amino acid oxidases with “narrow” or “strict” substrate specificity represent as good candidates to obtain an enzyme more suitable for biotechnological applications by enlarging their substrate specificity by means of protein engineering.     

Apoplastic oxidation of L-asparagine is involved in the control of the green algal endophyte Acrochaete operculata Correa & Nielsen by the red seaweed Chondrus crispus Stackhouse

Gametophytes of the marine alga Chondrus crispus are more resistant than tetrasporophytes to infection by the filamentous endophytic alga Acrochaete operculata. It has been shown recently that carrageenan oligosaccharides from the resistant gametophytic generation of C. crispus stimulate the secretion of L-asparagine (L-Asn) by the endophyte and that the host generates hydrogen peroxide and 2-oxo-succinamic acid after contact with this amino acid. Here the response of C. crispus to L-Asn and its effect on the pathogen is investigated. Chondrus crispus released hydrogen peroxide, ammonium ions, and a carbonyl compound into the medium when exposed to L-Asn. This response was correlated with an increase in oxygen consumption. Inhibitor studies indicated the involvement of a flavoenzyme in the reaction, which was sensitive to high concentrations of the reaction product, ammonium, and to chlorpromazine, quinacrine, and cyanide, inhibitors of L-amino acid oxidase. Cell wall macerate of C. crispus also responded to L-Asn, while protoplasts were inactive. Uptake of L-Asn into the cell was not necessary for the response, suggesting that the involved L-amino acid oxidase is apoplastic. Acrochaete operculata was more sensitive to hydrogen peroxide than C. crispus and settlement of A. operculata zoospores on C. crispus was reduced by 86% in the presence of L-Asn. This reduced settlement could be prevented with catalase. Chondrus crispus thus features an apoplastic amino acid oxidase, which is involved in the control of its endophytic pathogen. The modulation of the amino acid secretion in A. operculata by carrageenan oligosaccharides is therefore a key issue in the etiology of the association.     

L-氨基酸氧化酶的结构、底物谱、家族进化及应用研究进展

L-氨基酸氧化酶(LAAO)是一类生物体内参与氨基酸氧化代谢的重要氧化还原酶,能够以氧分子为电子受体催化L-氨基酸氧化脱氨,生成相应的酮酸、氨(NH₃)和过氧化氢(H₂O₂).近期发现有些LAAO能够专一性识别特定氨基酸,而不受其他种类氨基酸的干扰,因而在手性胺类化合物拆分、α-酮酸生物合成、临床样本、食品及氨基酸发酵过程中氨基酸含量检测等领域都发挥着重要作用.本文重点综述目前研究报道的底物专一性LAAO,总结并比较这些酶的酶学性质、结构功能,以及家族进化规律等,并进一步讨论这些酶在生物催化及氨基酸检测中的应用.本综述将为底物特异性LAAO的分子机制研究及产业应用研究提供重要的素材和指导.     

High-throughput Assays for Superoxide and Hydrogen Peroxide: DESIGN OF A SCREENING WORKFLOW TO IDENTIFY INHIBITORS OF NADPH OXIDASES*

Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate reader-based fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors. Using this newly developed approach along with the medium-throughput plate reader-based oximetry and EPR spin trapping as confirmatory assays, it is now eminently feasible to rapidly and reliably identify Nox enzyme inhibitors with a markedly lower rate of false positives. These methodological advances provide an opportunity to discover selective inhibitors of Nox isozymes, through enhanced conceptual understanding of their basic mechanisms of action.     

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.