Crystal Structure of Soybean Lipoxygenase L-1 at 1.4 Å Resolution<sup>,</sup>

Minor, Władek; Steczko, Janusz; Stec, Boguslaw; Otwinowski, Zbyszek; Bolin, Jeffrey T.; Walter, R.Erik; Axelrod, Bernard

doi:10.1021/bi960576u

Cited by 413 publications

(477 citation statements)

References 65 publications

(110 reference statements)

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“…However, CD/MCD spectroscopic data are more sensitive indicators of the first coordination sphere, as seen by the variation of the ligand arrangement depending on experimental conditions (28). While ligand bond lengths derived from crystallography may have to be viewed with caution, general structural features are represented reliably in the crystal structures; specifically, the hydrogen bond network connecting the first and second coordination spheres is welldefined in all known mammalian and plant LOs (24)(25)(26)(27). In this study, the correlation of Fe II ligand field spectroscopic data with structural information has allowed us to investigate the roles individual interactions within the H-bond network play in the modulation of enzyme reactivity in LOs.…”

Section: Discussionmentioning

confidence: 99%

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Spectroscopic Characterization of Soybean Lipoxygenase-1 Mutants: the Role of Second Coordination Sphere Residues in the Regulation of Enzyme Activity

et al. 2003

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Lipoxygenases are non-heme iron enzymes, which catalyze the stereo-and regiospecific hydroperoxidation of unsaturated fatty acids. Spectroscopic studies on soybean lipoxygenase have shown that the ferrous form of the enzyme is a mixture of five-and six-coordinate species (40 and 60%, respectively). Addition of substrate leads to a purely six-coordinate form. A series of mutations in the second coordination sphere (Q697E, Q697N, Q495A, and Q495E) were generated, and the structures of the mutants were solved by crystallography [Tomchick et al. (2001) Biochemistry 40, 7509-7517]. While this study clearly showed the contribution of H-bond interactions between the first and the second coordination spheres in catalysis, no correlation with the coordination environment of the Fe II was observed. A recent study using density-functional theory [Lehnert and Solomon (2002) J. Biol. Inorg. Chem. 8, 294-305] indicated that coordination flexibility, involving the Asn694 ligand, is regulated via H-bond interactions. In this paper, we investigate the solution structures of the second coordination sphere mutants using CD and MCD spectroscopy since these techniques are more sensitive indicators of the first coordination sphere ligation of Fe II systems. Our data demonstrate that the iron coordination environment directly relates to activity, with the mutations that have the ability to form a five-coordinate/six-coordinate mixture being more active. We propose that the H-bond between the weak Asn694 ligand and the Gln697 plays a key role in the modulation of the coordination flexibility of Asn694, and thus, is crucial for the regulation of enzyme reactivity.Lipoxygenases (LOs) 1 are mononuclear non-heme iron dioxygenases that catalyze the stereo-and regiospecific hydroperoxidations of cis,cis-1,4 polyunsaturated pentadienyl containing fatty acids. LOs have been isolated and characterized from numerous mammalian and plant sources and are believed to occur ubiquitously throughout these two phyla (1-3). In mammalian organisms, there are multiple LOs with differing regio-and stereospecificity against their substrate, arachidonic acid (AA). The products of these reactions are further metabolized by an array of enzymes leading to the formation of two major classes of cell effector molecules, leukotrienes and lipoxins, which are mediators in anaphylactic and inflammatory disorders (4, 5). Additionally, LO products are involved in a variety of metabolic functions, including organelle degradation (6), transcription regulation (7), and possibly tumor cell metastasis (8). For plant LOs, the main substrates are linoleic acid (LA) and linolenic acid, which are further metabolized to plant hormones, such as jasmonic acid. These hormones are involved in a variety of metabolic functions including plant growth and development, senescence, and cellular response to wounding and infections by pathogens (1). Soybeans have three LO isoforms (sLO-1, sLO-2, and sLO-3), which have homologous sequences and molecular weights of ∼96 kDa (9). This...

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Section: Discussionmentioning

confidence: 99%

“…Crystal structures for ferrous LOs have been solved for two soybean isoforms (sLO-1 and sLO-3) (14,(24)(25)(26) and rabbit reticulocyte 15-LO (15-rLO) (27). The three structures display a similar overall folding pattern.…”

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confidence: 99%

Spectroscopic Characterization of Soybean Lipoxygenase-1 Mutants: the Role of Second Coordination Sphere Residues in the Regulation of Enzyme Activity

et al. 2003

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“…The Nterminal amino acid analysis of mini-LOX showed the sequence STPIEFHSFQ, which corresponds to a unique trypsin cleavage site between lysine 277 and serine 278 (Figure 2A). Such a cleavage should indeed remove a ∼30 kDa N-terminal domain of LOX-1, leading to a fragment of the expected molecular mass of 63 695 Da (9,10,19). These data are consistent with the electrophoretic ( Figure 1A) and chromatographic ( Figure 1B) properties of mini-LOX.…”

Section: Generation Separation and Characterization Of Mini-loxmentioning

confidence: 99%

“…Mammalian lipoxygenases do not have the N-terminal domain present in LOX-1 and other related plant lipoxygenases, thus showing smaller molecular mass (75-80 kDa compared to 94-104 kDa in plants). It has been suggested that the N-terminal domain in LOX-1 makes only a loose contact with the C-terminal domain (9), and that it may be also dispensable for plant lipoxygenases (10), because all the amino acid side chains responsible for catalysis are located in the C-terminal domain. However, limited proteolysis experiments indicated that the two domains are instead tightly associated (20), and that domain-domain interactions play a role in the reversible unfolding of LOX-1 (21,22), through ionic interactions (13).…”

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Tryptic Digestion of Soybean Lipoxygenase-1 Generates a 60 kDa Fragment with Improved Activity and Membrane Binding Ability

et al. 2001

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Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a ∼30 kDa N-terminal domain and a ∼60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a ∼60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of R-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 × 10 6 and 10.9 × 10 6 M -1 ‚s -1 , respectively), the activation energy of the reaction being 4.5 ( 0.5 and 8.3 ( 0.9 kJ‚mol -1 for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, R-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio-and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inihibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio-(patho)logical conditions.Lipoxygenases are a family of monomeric non-heme, nonsulfur iron dioxygenases which catalyze the conversion of unsaturated fatty acids into conjugated hydroperoxides. Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory conditions such as arthritis, psoriasis, and bronchial asthma (1). They have been also implicated in atherosclerosis (2), brain aging (3), HIV infection (4), and kidney disease (5, 6). In plants, lipoxygenases favor germination and participate in the synthesis of traumatin and jasmonic acid and in the response to abiotic stress (7). Recently, they have been also shown to initiate the programmed death (apoptosis) of plant cells (8). Soybean lipoxygenase-1 (LOX-1) 1 is widely used as a prototype for studying the homologous family of lipoxygenases from tissues of different species, both in structural (9-13) and in kinetic (14-18) investigations. The amino acid sequence (19) and three-dimensional structure (9, 10) of LOX-1 have been determined. The overall 839 amino acid residues of LOX-1, with a molecular mass of 93 840 Da, show 2 domains. The first 146 N-terminal amino acid residues form an 8-stranded -barrel, and the remaining 693 residues of the C-terminal domain are organized into 23 helices and 8 -strands. Mammalian lipoxygenases do ...

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“…The crystal structures of soybean LOX-1 12,13 and LOX-3. 14,15 and that of a rabbit 15-LOX-1-inhibitor complex 16 have been reported so far.…”

Section: Introductionmentioning

confidence: 99%

Structural Stability of Soybean Lipoxygenase-1 in Solution as Probed by Small Angle X-ray Scattering

Dainese

Sabatucci

Zadelhoff

et al. 2005

Journal of Molecular Biology

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Soybean lipoxygenase-1 (LOX-1) is used widely as a model for studying the structural and functional properties of the homologous family of lipoxygenases. The crystallographic structure revealed that LOX-1 is organized in a b-sheet N-terminal domain and a larger, mostly helical, C-terminal domain. Here, we describe the overall structural characterization of native unliganded LOX-1 in solution, using small angle X-ray scattering (SAXS). We show that the scattering pattern of the unliganded enzyme in solution does not display any significant difference compared with that calculated from the crystal structure, and that models of the overall shape of the protein calculated ab initio from the SAXS pattern provide a close envelope to the crystal structure. These data, demonstrating that LOX-1 has a compact structure also in solution, rule out any major motional flexibility of the LOX-1 molecule in aqueous solutions. In addition we show that eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase used to mimic the effect of substrate binding, does not alter the overall conformation of LOX-1 nor its ability to bind to membranes. In contrast, the addition of glycerol (to 5%, v/v) causes an increase in the binding of the enzyme to membranes without altering its catalytic efficiency towards linoleic acid nor its SAXS pattern, suggesting that the global conformation of the enzyme is unaffected. Therefore, the compact structure determined in the crystal appears to be essentially preserved in these various solution conditions. During the preparation of this article, a paper by M. Hammel and co-workers showed instead a sharp difference between crystal and solution conformations of rabbit 15-LOX-1. The possible cause of this difference might be the presence of oligomers in the rabbit lipoxygenase preparations.

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Crystal Structure of Soybean Lipoxygenase L-1 at 1.4 Å Resolution^,

Cited by 413 publications

References 65 publications

Spectroscopic Characterization of Soybean Lipoxygenase-1 Mutants: the Role of Second Coordination Sphere Residues in the Regulation of Enzyme Activity

Spectroscopic Characterization of Soybean Lipoxygenase-1 Mutants: the Role of Second Coordination Sphere Residues in the Regulation of Enzyme Activity

Tryptic Digestion of Soybean Lipoxygenase-1 Generates a 60 kDa Fragment with Improved Activity and Membrane Binding Ability

Structural Stability of Soybean Lipoxygenase-1 in Solution as Probed by Small Angle X-ray Scattering

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Crystal Structure of Soybean Lipoxygenase L-1 at 1.4 Å Resolution,

Cited by 413 publications

References 65 publications

Spectroscopic Characterization of Soybean Lipoxygenase-1 Mutants: the Role of Second Coordination Sphere Residues in the Regulation of Enzyme Activity

Spectroscopic Characterization of Soybean Lipoxygenase-1 Mutants: the Role of Second Coordination Sphere Residues in the Regulation of Enzyme Activity

Tryptic Digestion of Soybean Lipoxygenase-1 Generates a 60 kDa Fragment with Improved Activity and Membrane Binding Ability

Structural Stability of Soybean Lipoxygenase-1 in Solution as Probed by Small Angle X-ray Scattering

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Crystal Structure of Soybean Lipoxygenase L-1 at 1.4 Å Resolution^,