Purification and characterization of a lentil seedling lipoxygenase expressed in E. coli: Implications for the mechanism of oxodiene formation by lipoxygenases

Hilbers, Martin P.; Finazzi‐Agrò, Alessandro; Veldink, Gerrit A.; Vliegenthart, Johannes F.G.

doi:10.1016/1357-2725(96)00018-0

Cited by 18 publications

(6 citation statements)

References 32 publications

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“…[20] and lentil LOX-1 [21], and for stress-induced LOX from potato [3] and rice [22]. This should enable their production on a scale for detailed biochemical and spectroscopic analyses, but all except the soya bean LOX are poorly characterized and, in addition, several have been expressed as fusion proteins, or are derived from cDNA clones that are not full-length.…”

Section: Introductionmentioning

confidence: 99%

“…Carbonyl production has been described as an anaerobic reaction of soya bean LOX-1 [25] and pea-seed LOX [7]. Most workers, however, studying native LOX from seeds of pea [6,[8][9][10], chickpea [26,27], broad bean [28] and kidney bean [10], from seedlings of lentil [21] and from rabbit reticulocytes [29] have shown that carbonyl production can take place aerobically. The source of the carbonyls is unclear, but they have been explained by the dissociation from LOX of the fatty acid radical following hydroperoxide activation and binding of linoleic acid [10], or the alkoxyl radical immediately following hydroperoxide activation [9].…”

Section: Introductionmentioning

confidence: 99%

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Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms

Hughes

Robinson

et al. 1998

Biochemical Journal

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The two major isoforms of lipoxygenase (LOX-2 and LOX-3) from pea (Pisum sativum L. cv. Birte) seeds have been cloned and expressed from full-length cDNAs as soluble, active, non-fusion proteins in Escherichia coli. A comparison of both isoforms purified to apparent homogeneity from E. coli and pea seeds has confirmed the authenticity of the recombinant products and established the properties of the native enzymes. Despite 86% similarity at the amino acid sequence level, the enzymes have distinct properties. They have been characterized in terms of specific activity, Fe content, optimum pH, substrate and product specificity, apparent Km and Vmax for the preferred substrate, linoleic acid, and interfacial behaviour with linoleic acid. We have used this evidence, in addition to EPR spectroscopy of the hydroperoxide-activated enzymes and estimates of kcat/Km, to propose different reaction mechanisms for linoleic acid oxidation for the two isoforms. The differences relate primarily to carbonyl production from linoleic acid for which we propose a mechanism. This implicates the release of a peroxyl radical in an aerobic hydroperoxidase reaction, as the source of the carbonyl compounds formed by dismutation of the liberated peroxyl radical.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms

Hughes

Robinson

et al. 1998

Biochemical Journal

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“…Lentil ( Lens culinaris ), a member of the Fabaceae, is an annual legume crop of nutritional quality higher than that of cereals, meat and fish, and is severely affected by pathogens [30]. Lentil seedlings contain different lipoxygenase (LOX) isozymes and we have characterized [31], cloned [32] and expressed in Escherichia coli [33] the isozyme most abundant in shoots. Lentil roots contain a different LOX, which shares several properties (e.g.…”

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confidence: 99%

Early activation of lipoxygenase in lentil (Lens culinaris) root protoplasts by oxidative stress induces programmed cell death

Maccarrone

Zadelhoff

Veldink

et al. 2000

European Journal of Biochemistry

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Oxidative stress caused by hydrogen peroxide (H 2 O 2 ) triggers the hypersensitive response of plants to pathogens. Here, short pulses of H 2 O 2 are shown to cause death of lentil (Lens culinaris) root protoplasts. Dead cells showed DNA fragmentation and ladder formation, typical hallmarks of apoptosis (programmed cell death). DNA damage was evident 12 h after the H 2 O 2 pulse and reached a maximum 12 h later. The commitment of cells to apoptosis caused by H 2 O 2 was characterized by an early increase of lipoxygenase activity, of ultraweak luminescence and of membrane lipid peroxidation, which reached 720, 350 and 300% of controls, respectively, at 6 h after H 2 O 2 treatment. Increased lipoxygenase activity was paralleled by an increase of its protein and mRNA level. Lipoxygenase inhibitors nordihydroguaiaretic acid, eicosatetraynoic acid and plamitoyl ascorbate prevented H 2 O 2 -induced DNA fragmentation and ultraweak luminescence, only when added together with H 2 O 2 , but not when added 8 h afterwards. Inhibitory anti-lipoxygenase monoclonal antibodies, introduced into the protoplasts by electroporation, protected cells against H 2 O 2 -induced apoptosis. On the other hand, lentil lipoxygenase products 9-and 13-hydroperoxy-octadecadienoic acids and their reduced alcohol derivatives were able to force the protoplasts into apoptosis. Altogether, these findings suggest that early activation of lipoxygenase is a key element in the execution of apoptosis induced by oxidative stress in plant cells, in a way surprisingly similar to that observed in animal cells.

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“…25 Concomitant formation of oxo-octadecadienoic acid (OXOD) was estimated by measuring absorbance at 268 nm (ε = 22,000 cm…”

Section: Resultsmentioning

confidence: 99%

A Substrate Serves as a Hydrogen Atom Donor in the Enzyme-Initiated Catalytic Mechanism of Dual Positional Specific Maize Lipoxygenase-1

Huon¹,

Jang²,

Cho³

et al. 2009

Bulletin of the Korean Chemical Society

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The maize lipoxgyenase-1 is a non-traditional dual positional specific enzyme and the reaction proceeds via enzyme-initiated catalysis. Bioinformatic analysis indicated that the maize lipoxygenase-1 is structurally more similar to soybean LOX1 than pea LOXN2 in that it has an additional external loop (residues 318-351) in the carboxy-terminal catalytic domain. We analyzed the dependence of product distribution on concentration of linoleic acid and monitored the formation of hydroperoxyoctadecadienoic acid as a function of enzyme concentration. Product distribution was strongly influenced by substrate concentration, such that kinetically-controlled regioisomers were enriched and thermodynamically-controlled regioisomers were depleted at high substrate concentration. Kinetic studies indicated that the formation of hydroperoxyoctadecadienoic acid saturated rapidly in an enzyme concentration-dependent manner, which implied that reactivation by reoxidation of inactive Fe(II) failed to occur. Our results support the previously proposed enzyme-initiated catalytic mechanism of the maize lipoxgyenase-1 and reveals that a substrate molecule serves as a hydrogen atom donor in its enzyme-initiated catalysis.

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Purification and characterization of a lentil seedling lipoxygenase expressed in E. coli: Implications for the mechanism of oxodiene formation by lipoxygenases

Cited by 18 publications

References 32 publications

Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms

Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms

Early activation of lipoxygenase in lentil (Lens culinaris) root protoplasts by oxidative stress induces programmed cell death

A Substrate Serves as a Hydrogen Atom Donor in the Enzyme-Initiated Catalytic Mechanism of Dual Positional Specific Maize Lipoxygenase-1

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