The antimicrobial activity of marinocine, synthesized by Marinomonas mediterranea, is due to hydrogen peroxide generated by its lysine oxidase activity

Marinocine is a broad-spectrum antibacterial protein synthesized by the melanogenic marine bacterium Marinomonas mediterranea. This work describes the basis for the antibacterial activity of marinocine and the identification of the gene coding for this protein. The antibacterial activity is inhibited under anaerobic conditions and by the presence of catalase under aerobic conditions. Marinocine is active only in culture media containing l-lysine. In the presence of this amino acid, marinocine generates hydrogen peroxide, which causes cell death as confirmed by the increased sensitivity to marinocine of Escherichia coli strains mutated in catalase activity. The gene coding for this novel enzyme was cloned using degenerate PCR with primers designed based on conserved regions in the antimicrobial protein AlpP, synthesized by Pseudoalteromonas tunicata, and some hypothetical proteins. The gene coding for marinocine has been named lodA, standing for lysine oxidase, and it seems to form part of an operon with a second gene, lodB, that codes for a putative dehydrogenase flavoprotein. The identity of marinocine as LodA has been demonstrated by N-terminal sequencing of purified marinocine and generation of lodA mutants that lose their antimicrobial activity. This is the first report on a bacterial lysine oxidase activity and the first time that a gene encoding this activity has been cloned.     

Complete genome sequence of the melanogenic marine bacterium Marinomonas mediterranea type strain (MMB-1(T))

Marinomonas mediterranea MMB-1(T) Solano & Sanchez-Amat 1999 belongs to the family Oceanospirillaceae within the phylum Proteobacteria. This species is of interest because it is the only species described in the genus Marinomonas to date that can synthesize melanin pigments, which is mediated by the activity of a tyrosinase. M. mediterranea expresses other oxidases of biotechnological interest, such as a multicopper oxidase with laccase activity and a novel L-lysine-epsilon-oxidase. The 4,684,316 bp long genome harbors 4,228 protein-coding genes and 98 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Deamination of protein lysyl ε-amino groups by peroxidase in vitro

Peroxidase, catechol, and hydrogen peroxide were shown to react with proteins, causing a decrease in lysine detectable after acid hydrolysis. The loss of lysine did not occur in the presence of benzenesulfinic acid which suggested that the quinones formed by peroxidase had oxidized some lysyl residues to lysyl aldehyde that formed a cyclic ninhydrin negative Shiff's base. When peroxidase treated protein was oxidized with performic acid prior to hydrolysis a new ninhydrin positive compound was found, which was shown by cochromatography and mass spectroscopy to be α-aminoadipic acid. The α-aminoadipic acid recovered accounted for (20–40)% of the lysine lost.     

Hydrogen peroxide linked to lysine oxidase activity facilitates biofilm differentiation and dispersal in several gram-negative bacteria

The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP, which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal, and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death. We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays an important role in dispersal and colonization across a range of gram-negative bacteria.     

Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea

This work describes the purification and partial characterization of a novel antibacterial compound, here named marinocine, produced by Marinomonas mediterranea, a melanogenic marine bacterium with rich secondary metabolism. The antibacterial compound is a protein detected in the medium at death phase of growth. It has been purified to apparent homogeneity from the supernatants of cultures by means of ethanol precipitation followed by column chromatographies on DEAE-Sephadex and Sephacryl HR-200. The protein has an apparent molecular mass of 140-170 kDa according to gel permeation chromatography and non-denaturing SDS-PAGE, although in denaturing SDS-PAGE two mayor bands of 97 and 185 kDa appear. Marinocine is relatively heat-stable and shows a great resistance against many hydrolytic enzymes such as glycosidases, lipase, and proteases. The antibacterial range of the molecule includes Gram-positive and Gram-negative microorganisms, as well as some nosocomial isolates, Staphylococcus aureus and Pseudomonas sp., highly resistant to classical antibiotics. By contrast, marinocine did not show any effect on the eukaryotic microorganisms tested. Regarding eukaryotic CHO cells, the decrease on viability was much lower than the one observed on bacterial cells.     

Lysine is catabolized to 2-aminoadipic acid in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> by an omega-aminotransferase and to saccharopine by a lysine 2-ketoglutarate reductase. Characterization of the omega-aminotransferase

The biosynthesis and catabolism of lysine in Penicillium chrysogenum is of great interest because these pathways provide 2-aminoadipic acid, a precursor of the tripeptide δ-L-2-aminoadipyl-L-cysteinyl-D-valine that is an intermediate in penicillin biosynthesis. In vivo conversion of labelled L-lysine into two different intermediates was demonstrated by HPLC analysis of the intracellular amino acid pool. L-lysine is catabolized to 2-aminoadipic acid by an ω-aminotransferase and to saccharopine by a lysine-2-ketoglutarate reductase. In lysine-containing medium both activities were expressed at high levels, but the ω-aminotransferase activity, in particular, decreased sharply when ammonium was used as the nitrogen source. The ω-aminotransferase was partially purified, and found to accept L-lysine, L-ornithine and, to a lesser extent, N-acetyl-L-lysine as amino-group donors. 2-Ketoglutarate, 2-ketoadipate and, to a lesser extent, pyruvate served as amino group acceptors. This pattern suggests that this enzyme, previously designated as a lysine-6-aminotransferase, is actually an ω-aminotransferase. When 2-ketoadipate is used as substrate, the reaction product is 2-aminoadipic acid, which contributes to the pool of this intermediate available for penicillin biosynthesis. The N-terminal end of the purified 45-kDa ω-aminotransferase was sequenced and was found to be similar to the corresponding segment of the OAT1 protein of Emericella (Aspergillus) nidulans. This information was used to clone the gene encoding this enzyme.     

Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis.

Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-14C]lysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C]saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed.     

The effect of L-lysine alpha-oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D on the rat pheochromocytoma PC12 cell line

L-Amino acid oxidases (L-AAO; EC 1.4.3.2) comprise a group of flavoproteins that catalyze oxidative deamination of L-alpha amino acids to corresponding alpha-keto acids, NH₃ and H₂O₂. Most of these enzymes are homodimers with molecular mass of 100–150 kDa that exhibit antiviral, antifungal, antibacterial, and anticancer activity. Among this group of enzymes L-lysine alpha-oxidase (LO) is especially important as its biological effects may differ from the effects of other L-AAO, because this enzyme preferentially oxidizes L-lysine, the essential amino acid for the human body, without any practical effect on other amino acids. Since molecular mechanisms of the cytotoxic action of LO still require better understanding, in this study we have investigated a possible mechanism of action of LO from Trichoderma cf. aureoviride Rifai VKMF-4268D. A rat pheochromocytoma PC12 cell culture was used as a model. Using flow cytometry a dose-dependent cell death induced by LO was shown. The increase in intracellular reactive oxygen species detected by the 2,7-dichlorodihydrofluorescein assay suggests that the oxidative pathway is one of mechanisms underlying the cytotoxic LO action; however, this does not rule out the involvement of other (previously demonstrated) mechanisms of LO effects on cell death.     

D-氨基酸氧化酶研究进展

D-氨基酸氧化酶(D-amino acid oxidase:oxidoreductase, DAAO, EC 1.4.3.3)是一种以黄素腺嘌呤(FAD)为辅基的典型黄素蛋白酶类,可氧化D-氨基酸的氨基生成相应的酮酸和氨。在体内D-氨基酸的代谢中起着重要作用。主要介绍了D-氨基酸氧化酶的生理功能和应用、表达条件优化及通过定点突变对酶学性质的研究。     

Preventive effect of tannic acid on 2-acetylaminofluorene induced antioxidant level, tumor promotion and hepatotoxicity: a chemopreventive study

Tannic acid, present in almost every food derived from plants, has been widely investigated as a chemopreventive agent because, apart from its use as a food additive, pharmacological studies have demonstrated its many health-promoting properties. In this study, we show the modulatory effect of tannic acid on 2-acetylaminofluorene (2-AAF)-mediated hepatic oxidative stress and cell proliferation in rats. 2-AAF (50 mg/kg body weight) caused reduction in hepatic glutathione content and the activities of hepatic anti-oxidant enzymes and phase-II metabolizing enzymes with an enhancement of xanthine oxidase activity, lipid peroxidation and hydrogen peroxide content. 2-AAF treatment also induced serum oxaloacetate and pyruvate transaminase, lactate dehydrogenase and gamma-glutamyl transpeptidase. Treatment of rats orally with tannic acid (125 and 250 mg/kg body weight) resulted in significant recovery of hepatic glutathione content, antioxidant and phase-II metabolizing enzymes. Also, significant decreases in lipid peroxidation, xanthine oxidase, hydrogen peroxide generation and liver damage marker enzymes were observed. The antiproliferative efficacy of the tannic acid was also evaluated. The promotion parameters induced (ornithine decarboxylase activity and DNA synthesis) by 2-AAF administration in the diet with partial hepatectomy (PH) were also significantly suppressed, dose dependently, by tannic acid. Hence, we propose that tannic acid might suppress the promotion stage via inhibition of oxidative stress and polyamine biosynthetic pathway.     

单胺氧化酶

单胺氧化酶（monoamine oxidase,MAO）是生物体内一种十分重要的酶,它在大脑和周围神经组织中催化一些生物体产生的胺,氧化脱氨产生过氧化氢（H₂O₂）.单胺氧化酶A和B基因的克隆清楚地证明了这些酶是由不同的多肽组成的.单胺氧化酶A和B的基因定位于X染色体（Xp11.23）,都由15个外显子组成,而且它们的内含子-外显子组织是完全一致的.这些事实表明单胺氧化酶A和B的基因很可能从同一个祖先进化而来.单胺氧化酶A和B具有不同的底物和抑制剂专一性,在生物神经递质代谢和行为方面具有不同的作用.     

Pipecolic acid biosynthesis in Rhizoctonia leguminicola. II. Saccharopine oxidase: a unique flavin enzyme involved in pipecolic acid biosynthesis

The fungal parasite Rhizoctonia leguminicola produces two indolizidine alkaloids, slaframine and swainsonine, of physiological interest. These alkaloids are biosynthesized from pipecolic acid which in turn is derived from L-lysine in this fungus as shown in the accompanying paper (Wickwire, B.M., Harris, C.M., Harris, T.M., and Broquist, H.P. (1989) J. Biol. Chem. 265, 14742-14747): L-lysine----saccharopine----delta 1----piperideine-6- carboxylate----pipecolate. This paper concerns the discovery, purification, and properties of a flavoenzyme, termed saccharopine oxidase, which carries out the oxidative cleavage of saccharopine as follows: Saccharopine + O2----delta 1-piperidine-6-carboxylate + glutamate + H2O2 The enzyme was purified 2,000-fold to homogeneity (polyacrylamide gel electrophoresis) in 14% yield from R. leguminicola mycelia, and had a native molecular mass of about 45,000 daltons by gel filtration (fast protein liquid chromatography Superose). Evidence for the presence of a flavin in the enzyme was drawn from these considerations: (a) the enzyme, while oxidatively cleaving saccharopine, concomitantly reduces 2,6-dichlorophenolindophenol; (b) the purified enzyme has a fluorescence spectrum typical of flavins; and (c) the enzyme requires oxygen and produces hydrogen peroxide. Good correlation was shown with purified saccharopine oxidase between disappearance of saccharopine with the concomitant appearance of delta 1-piperideine-6-carboxylate plus glutamate. The enzyme has a pH optimum about 6 and a Km for saccharopine of 0.128 mM. The enzyme apparently exists in R. leguminicola to shunt saccharopine, a major lysine metabolite, into a secondary pathway of lysine metabolism leading to pipecolate and subsequently to slaframine and swainsonine.     

Expression of active spinach glycolate oxidase in Aspergillus nidulans

The biocatalytic production of glyoxylic acid from glycolic acid requires two enzymes: glycolate oxidase, which catalyzes the oxidation of glycolic acid by oxygen to produce glyoxylic acid and hydrogen peroxide, and catalase, which decomposes the byproduct hydrogen peroxide. As an alternative to isolation from the leaf peroxisomes of spinach, glycolate oxidase has now been cloned and expressed in transformants of Aspergillus nidulans T580 at levels ranging from 1.7 to 36 IU/g dry wt. cells. The glycolate oxidase of transformant strain T17 comprises ca. 1.9% of total cell protein and is expressed at near 100% activity. (c) 1996 John Wiley & Sons, Inc.     

A possible involvement of sulfhydryl groups in the conversion of lysine monooxygenase to an oxidase.

Lysine monooxygenase catalyzes the oxygenation of lysine and arginine, and produces delta-amino-n-valeramide and gamma-guanidinobutyramide, respectively, concomitant with decarboxylation. In a preliminary communication, treatment of the native enzyme with p-chloromercuribenzoate was shown to inactivate the oxygenase and to induce an oxidase activity. The modified enzyme catalyzed predominantly the oxidative deamination of lysine and arginine resulting in the formation of the corresponding alpha-keto acid, ammonia, and hydrogen peroxide (YAMAUCHI, T., YAMAMOTO, S., and HAYAISHI, O.(1973) J. Biol. Chem. 2j8, 3750-3752). Paper electrophoresis, cellulose thin layer chromatography, and chemical degradation of the reaction products from lysine and arginine, provided further evidence for their identity with alpha-keto-epsilon-aminocaproate and alpha-keto-delta-guanidinovalerate, respectively. Further studies were carried out to establish the involvement of sulfhydryl groups in this conversion of the enzyme activities. Various sulfhydryl reagents including certain mercurials, alkylating, and oxidizing reagents, showed essentially identical effects on the enzyme. Dithiothreitol treatment reversed the conversion produced by various mercurials; the oxidase activity disappeared and the oxygenase activity was recovered. When p-chloromercuribenzoate was added to the enzyme and the increase in the absorbance at 250 nm was followed, 3.6 of the 6.5 half-cystine residues present per enzyme-bound FAD were readily titrated within 3 to 4 min. The inactivation of the oxygenase and the induction of the oxidase activity were almost maximal with 4 to 5 mol of p-chloromercuribenzoate/mol of enzyme, and these effects occurred within 3 to 4 min. These results together with other properties of the modified enzyme provided evidence for a possible involvement of these reactive sulfhydryl groups during the conversion of the oxygenase to an oxidase.     

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.     

New biotech applications from evolved D-amino acid oxidases

D-Amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxygen-dependent oxidative deamination of amino acid D-isomers with absolute stereospecificity, which results in α-keto acids, ammonia and hydrogen peroxide. Recently, the extraordinary functional plasticity of DAAO has become evident; in turn, boosting research on this flavoprotein. Protein engineering has allowed for a redesign of DAAO substrate specificity, oxygen affinity, cofactor binding, stability, and oligomeric state. We review recent developments in utilizing DAAO, including as a biocatalyst for resolving racemic amino acid mixtures, as a tool for biosensing, and as a new mechanism of herbicide resistance. Perspectives for future biotechnological applications of this oxidative biocatalyst are also outlined.     

Modification of Proteins in Endothelial Cell Death during Oxidative Stress

Exposure of bovine aortic endothelial cells in vitro to oxidative stress causes a cascade of changes in cell function, culminating in cell death if the stress is sufficiently severe. Oxidative modification of proteins, as measured by the reaction of 2,4-dinitrophenylhydrazine with carbonyl groups of oxidized proteins, increased three- to fourfold in endothelial cells exposed to hydrogen peroxide or to a xanthine/xanthine oxidase system. The increase in oxidative modification of protein occurred rapidly, preceding loss of cellular ATP and eventual cell death. Oxidative modification of protein was paralleled by loss of activity of the key metabolic enzymes, glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. The finding that oxidative modification of protein is an early event following oxidative stress suggests that oxidative modification of protein is not only a marker for oxidative damage but also a causal factor in oxidative injury. Published by Elsevier Science Inc.     

Amine oxidase-like activity of polyphenols. Mechanism and properties.

Polyphenols in several oxidation systems gained amine oxidase-like activity, probably due to the formation of the corresponding quinones. In the presence of Cu(II), o- and p-phenolic compounds exhibited amine oxidase-like activity, whereas only the o-phenolic compounds showed the activity in the presence of 1,1-diphenyl-2-picrylhydrazyl radical. The activity was determined by measuring the conversion of benzylamine to benzaldehyde by HPLC. Moreover, gallic acid, chlorogenic acid, and caffeic acid, which are plant polyphenols, converted the lysine residue of bovine serum albumin to alpha-amino-adipic semialdehyde residue, indicating lysyl oxidase-like activity. We also characterized the activity of pyrocatechol, hydroquinone, and pyrogallol in the presence of Cu(II). The oxidative deamination was accelerated at a higher pH, and required O2 and transition metal ions. Furthermore, EDTA markedly inhibited the reaction but not beta-aminopropionitrile, which is a specific inhibitor of lysyl oxidase. Catalase significantly inhibited the oxidation, implying the participation of hydroxyl radical in the reaction, but superoxide dismutase stimulated the oxidation, probably due to its radical formation activity. We discussed the mechanism of the oxidative deamination by polyphenols and the possible significance of the activity for biological systems.     

Attenuation of oxidative stress, inflammation and early markers of tumor promotion by caffeic acid in Fe-NTA exposed kidneys of Wistar rats

Iron nitrilotriacetate (Fe-NTA), a chief environmental pollutant, is known for its extensive toxic manifestations on renal system. In the present study, caffeic acid, one of the most frequently occurring phenolic acids in fruits, grains, and dietary supplements was evaluated for its shielding effect against the Fe-NTA-induced oxidative, inflammatory, and pathological damage in kidney. Fe-NTA was administered (9 mg Fe/kg body weight) intraperitoneally to the Wistar male rats on 20th day while caffeic acid was administered orally (20 and 40 mg/kg body weight) before administration of Fe-NTA. The intraperitoneal administration of Fe-NTA-enhanced lipid peroxidation, xanthine oxidase, and hydrogen peroxide generation with reduction in renal glutathione content, antioxidant enzymes, viz., catalase, glutathione peroxidase, and glutathione reductase. A sharp elevation in the levels of myloperoxidase, blood urea nitrogen (BUN), and serum creatinine has also been observed. Tumor promotion markers viz., ornithine decarboxylase (ODC) and [(3)H] thymidine incorporation into renal DNA were also significantly increased. Treatment of rats orally with caffeic acid (20 and 40 mg/kg body weight) resulted in a significant decrease in xanthine oxidase (P < 0.001), lipid peroxidation (P < 0.001), γ-glutamyl transpeptidase (P < 0.01), and H(2)O(2) (P < 0.01). There was significant recovery of renal glutathione content (P < 0.001) and antioxidant enzymes (P < 0.001). There was also a reversal in the enhancement of renal ODC activity, DNA synthesis, BUN, and serum creatinine (P < 0.001). All these changes were supported by histological observations. The results indicate that caffeic acid may be beneficial in ameliorating the Fe-NTA-induced oxidative damage and tumor promotion in the kidney of rats.     

The role of cationized catalase and cationized glucose oxidase in mucosal oxidative damage induced in the rat jejunum.

The successful prevention of hydrogen peroxide-induced damage to the rat jejunal mucosa by cationized catalase is described in this study. Biological damage was induced in a closed circulating intestinal loop of the rat by hydrogen peroxide and by hydroxyl radicals induced in situ via the metal-mediated Haber-Wiess reaction. The mucosal activity of lactate dehydrogenase and the amount of potassium ions were used to quantitatively characterize the tissue damage. Catalase was cationized by reacting it with N,N'-dimethyl-1,3-propanediamine to give a soluble product or with polyhistidine to give an insoluble product. The activity of the modified enzymes was assessed, and their ability to protect the rat jejunal mucosa against oxidative stress was studied. It was found that in all cases the cationized enzymes were superior to the native catalase in their shield capability. A significant protection against Fe(II)/H2O2 and ascorbic acid/copper ion-mediated damage was obtained when the cationized enzymes were used. In the presence of glucose, native glucose oxidase failed to cause damage in the rat jejunal mucosa; however, the cationized enzyme caused profound tissue injury. These findings indicate the potential therapeutic merit of cationized enzymes for the treatment of pathological processes in the intestine, whenever oxidative stress is involved.