Three-dimensional structure of a purple lipoxygenase.

Polyunsaturated fatty acid metabolism is governed primarily by two enzymes, prostaglandin H synthase and lipoxygenase. The crystal structure of the metastable product-oxidized purple form of soybean lipoxygenase-3 was determined at 2.0 A resolution. The data reveal that the chromophore corresponds to an iron-peroxide complex, a potential intermediate in the catalyzed reaction. A significant alteration of the iron site accompanies the formation of the complex. The structure, the first for a fatty acid-lipoxygenase complex, also reveals an unexpected mode of binding, and identifies amino acid residues that may play significant roles in catalysis, regio- and stereoselectivity.

Iron extraction from soybean lipoxygenase 3 and reconstitution of catalytic activity from the apoenzyme.

Lipoxygenases contain a unique nonheme iron cofactor with a redox role in the catalyzed reaction. The conditions for the extraction of the metal atom were investigated for one of the soybean lipoxygenase isoenzymes. Removal of the iron by o-phenanthroline was attained in the presence of substrate under anaerobic conditions, but the apoenzyme could not be isolated and reconstituted. The freshly regenerated sodium form of Chelex-100 also removes the iron atom from native soybean lipoxygenase 3, but only in sodium bicarbonate buffer at pH 8.0. The soluble but inactive apoenzyme was reconstituted with ferric ammonium sulfate in Tris--HCl buffer at pH 7.0. Stoichiometric iron in the reconstituted enzyme was established using inductively coupled plasma-atomic emission spectroscopy. The reconstituted enzyme contained 90 +/- 10% of the specific activity of the native enzyme. The native configuration of the reconstituted iron site was confirmed by electron paramagnetic resonance spectroscopy.

Structural and thermochemical characterization of lipoxygenase-catechol complexes.

A complex between native, iron(II) soybean lipoxygenase 3 and 4-nitrocatechol, a known inhibitor of the enzyme, has been detected by isothermal titration calorimetry and characterized by X-ray crystallography. The compound moors in the central cavity of the protein close to the essential iron atom, but not in a bonding arrangement with it. The iron ligands experience a significant rearrangement upon formation of the complex relative to their positions in the native enzyme; a water molecule becomes bound to iron in the complex, and one histidine ligand moves away from the iron to become involved in a hydrogen bonding interaction with the catechol. These changes in position result in a trigonal pyramid coordination geometry for iron in the complex. Molecular modeling and force field calculations predict more than one stable complex between 4-nitrocatechol and the central cavity of lipoxygenase 3, but the interaction having the small molecule in the same orientation as the one found in the crystal structure was the most favorable. These observations reveal specific details of the interaction between lipoxygenase and a small molecule and raise the possibility that changes in the ligand environment of the iron atom could be a feature of the product activation reaction or the catalytic mechanism.

Structure of soybean lipoxygenase L3 and a comparison with its L1 isoenzyme.

Soybean lipoxygenase isoenzyme L3 represents a second example (after L1) of the X-ray structure (R = 17% at 2.6 A resolution) for a member of the large family of lipoxygenases. L1 and L3 have different characteristics in catalysis, although they share 72% sequence identity (the changes impact 255 amino acids) and similar folding (average C alpha rms deviation of 1 A). The critical nonheme iron site has the same features as for L1:3O and 3N in pseudo C3v orientation, with two oxygen atoms (from Asn713 and water) at a nonbinding distance. Asn713 and His518 are strategically located at the junction of three cavities connecting the iron site with the molecule surface. The most visible differences between L1 and L3 isoenzymes occur in and near these cavities, affecting their accessibility and volume. Among the L1/L3 substitutions Glu256/ Thr274, Tyr409/His429, and Ser747/Asp766 affect the salt bridges (L1: Glu256...His248 and Asp490...Arg707) that in L1 restrict the access to the iron site from two opposite directions. The L3 molecule has a passage going through the whole length of the helical domain, starting at the interface with the Nt-domain (near 25-27 and 254-278) and going to the opposite end of the Ct-domain (near 367, 749). The substrate binding and the role of His513, His266, His776 (and other residues nearby) are illustrated and discussed by using models of linoleic acid binding. These hypotheses provide a possible explanation for a stringent stereo-specificity of catalytic products in L1 (that produces predominantly 13-hydroperoxide) versus the lack of such specificity in L3 (that turns out a mixture of 9- and 13-hydroperoxides and their diastereoisomers).

Flash-freezing causes a stress-induced modulation in a crystal structure of soybean lipoxygenase l3.

A dynamic conformational flexibility of a protein might be a source of non-covalent structural heterogeneity, causing diminished diffracting ability of crystals and disorder in a crystal structure of soybean lipoxygenase L3. Room-temperature data, space group C2, correspond to a structure with large channels lined mostly or in part by disordered fragments of the molecule or flexible loops with an increased thermal vibration. A rapid change in temperature of approximately 200 K creates a wave of a stress-induced modulation that propagates in the crystal changing its reciprocal space into a three-dimensional quilt-like mixture of C and P intertwined lattices. Low-temperature data indicate a transformation from the dynamic to static disorder, leading to a primitive unit cell with 10% reduced volume. The molecules, formerly related by a twofold axis are rotated by approximately 7 degrees and are shifted along the diagonal to be approximately 4 A, closer together. During a routine data collection for the flash-frozen crystals of similar properties such phenomena could easily go unnoticed leading to biased results because of such effects and possibly improper indexing of the data.

Catalysis sensitive conformational changes in soybean lipoxygenase revealed by limited proteolysis and monoclonal antibody experiments.

Soybean lipoxygenases catalyze lipid hydroperoxidation of polyunsaturated fatty acids. Putative ligand mediated conformational changes in soybean lipoxygenase 3 (L3) were studied by a combination of limited proteolysis and a series of monoclonal antibodies that recognize discontinuous epitopes and alter catalysis (inhibition and activation). Trypsin cleaved L3 (97 kDa) into C-terminal 60 kDa and N-terminal 37 kDa fragments. The 37 kDa fragment was obtained from a 38 kDa fragment formed initially. Using protein footprinting, the epitopes of the antibodies were mapped to the 37 kDa fragment. Proteolysis in the presence of a substrate analog inhibitor, oleic acid, generated the 60 and the 38 kDa fragments only. No further proteolysis of the 38 kDa fragment was seen even after prolonged incubation. This was not a detergent effect since the altered proteolysis was not obtained in the presence of SDS or Tween 20. Binding of a monoclonal antibody to L3 in the presence of oleic acid was substantially reduced providing additional evidence for a conformational change induced by the oleic acid-lipoxygenase interaction. These observations are interpreted using the recently solved three-dimensional structure of L3. It is apparent that while the protein is composed of a small N-terminal beta-barrel domain and a large principally alpha-helical C-terminal domain, proteolysis does not take place at a linking region between the two domains. The proteolysis results makes it clear that the smaller domain is connected across the entire length of the larger domain to a narrow, tongue-like projection that extends into the vicinity of the entrance to the proposed substrate binding channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Position 713 is critical for catalysis but not iron binding in soybean lipoxygenase 3.

The role of asparagine-713 in iron atom incorporation and catalysis in soybean lipoxygenase 3 was investigated using site-directed mutagenesis. A full-length cDNA for the lipoxygenase isoenzyme was obtained from a library derived from soybeans cv. Provar. Protein with native specific activity at pH 7.4 was obtained from expression in Escherichia coli. Two recent structure reports provided conflicting views about the participation of the side chain of asparagine-694 in the coordination of the iron atom required for catalysis by lipoxygenase 1. Oligonucleotide-directed mutagenesis was employed to modify residue 713 in lipoxygenase 3 which corresponds to asparagine-694 in the sequence of lipoxygenase 1. It was found that for enzyme expressed in bacteria, asparagine was not required for iron incorporation. Histidine, alanine, and serine substitutions for asparagine-713 all produced proteins that contained iron. The histidine mutant had specific activity and catalytic characteristics comparable to the wild-type enzyme. By contrast, the alanine- and serine-substituted lipoxygenases had no detectable catalytic activity. When oxidized by product, the histidine mutant also displayed the characteristic g6 signal of the soybean enzyme in its EPR spectrum. The possibilities that the residue at position 713 acts as a metal ligand, an acid-base catalyst, and a hydrogen bonding group are considered and discussed.

Rapid purification of rabbit reticulocyte lipoxygenase for electron paramagnetic spectroscopy characterization of the non-heme iron.

An efficient three-step purification technique has been developed for the reticulocyte 15-lipoxygenase from rabbit. Ammonium sulfate fractionated reticulocyte lysate was purified by size exclusion chromatography and preparative scale isoelectric focusing. The entire procedure was complete in less than eight hours and was carried out in batches which typically yielded 10 mg of purified enzyme. The identity and purity of the enzyme were evaluated by N-terminal sequencing, sodium dodecylsulfate polyacrylamide gel electrophoresis and specific activity determinations. The enzyme contained approximately one g-atom iron per mole of protein. The iron was present in an electron paramagnetic spectroscopy (EPR) silent, presumably high spin iron(II), form in the isolated enzyme. Treatment with one equivalent of 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid resulted in the appearance of an EPR signal around g6.

Limited proteolysis and active-site labeling studies of soybean lipoxygenase.

Soybean lipoxygenase 1 was studied using limited proteolysis and active-site labeling to begin the structural characterization of the enzyme in solution. The serine proteases trypsin and chymotrypsin cleaved the large monomeric protein (95 kDa) into two large polypeptides, a C-terminal fragment of about 30 kDa and an N-terminal fragment of about 60 kDa. Under conditions that led to complete cleavage of the protein as judged by SDS-polyacrylamide gel electrophoresis, the catalytic activity of the protein was either reduced slightly (chymotrypsin) or enhanced (trypsin). The characteristics of the cleaved enzymes were the same as for native lipoxygenase 1 in all aspects examined: insensitivity to cyanide, fluoride, and EDTA, regiochemical and stereochemical consequences of catalysis, and EPR spectroscopy upon oxidation by product. The two fragments apparently were tightly associated as they could not be resolved under conditions which preserved the catalytic activity. Both native and protease-cleaved lipoxygenase 1 formed covalent adducts when treated with either 14C-phenylhydrazine or 4-nitrophenylhydrazine. The label was found only in the 60-kDa fragment and following complete trypsin digestion was associated with a peptide beginning after Lys-482 in the primary sequence. Therefore labeling occurred in the vicinity of the conserved histidine cluster which has been postulated as the iron-binding site. From these observations it appears that lipoxygenase 1 exists as a pair of tightly associated domains with the iron-binding site located in the larger of the two.

Preparation and characterization of monoclonal antibodies against soybean seed lipoxygenase isoenzymes.

Sets of monoclonal antibodies have been prepared using two soybean seed lipoxygenase isoenzymes as the antigens. The antibodies were characterized by ELISA, Western blot analysis, immunoprecipitation, and in kinetic assays. Several antibodies displaying selectivity for the two closely related polypeptides were obtained, while the majority of the antibodies generated were crossreactive. Antibodies specific to the native and denatured forms of the two proteins were also obtained. Two of the monospecific antibodies were shown to immunoprecipitate the appropriate isoenzyme selectively from a mixture. When these antibodies were immobilized on agarose, they were successful in the immunoaffinity purification of the individual isoenzymes. In kinetic experiments certain antibodies were found to influence catalysis upon incubation with lipoxygenase. Antibodies which both inhibited and stimulated catalysis were identified.

The initial characterization of the iron environment in lipoxygenase by Mössbauer spectroscopy.

The incorporation of 57Fe into two lipoxygenase isoenzymes from soybeans has been achieved making possible the first observations of the iron environment in these proteins using Mössbauer spectroscopy. Immature soybean seeds were grown in tissue culture medium supplied with 57Fe. The iron in the active lipoxygenases that were isolated from the cultured seeds was readily detected in Mössbauer measurements. It was unequivocally demonstrated that the native enzyme contains high-spin Fe(II). Based on the sign of the electric field gradient, the most likely ligand sphere for the iron in native lipoxygenase consists of oxygen and nitrogen ligands in a roughly octahedral field of symmetry. It was possible to detect Mössbauer signals in highly concentrated samples of native lipoxygenases containing 57Fe at natural abundance. The spectra obtained for enriched and natural abundance native enzyme had the same high-spin Fe(II) Mössbauer parameters. This confirmed that the environment of the iron in enzymes isolated from cultured seeds and dry soybeans were the same. The Mössbauer spectra (4.2-250 K) for samples of both isoenzymes after oxidation of the iron in native enzyme by the product of lipoxygenase catalysis were extremely broad (20 mm/s) with no obvious narrow resonance lines. This was the result of the existence of paramagnetically broadened spectra for such samples even at relatively high temperature as evidenced by the appropriate EPR signal. A small molecule containing an iron site sharing many of these Mössbauer and electron paramagnetic resonance properties with lipoxygenase was identified: Fe(II)/(III).diethylenetriaminepentaacetic acid.

Crystallization and preliminary X-ray characterization of a soybean seed lipoxygenase.

An isoenzyme of soybean (Glycine max L. Merrill cv. Provar) lipoxygenase (EC 1.13.11.12) has been crystallized using the vapor diffusion method. Crystals were grown from solutions of the protein (7 mg/ml) using 10 to 20% (w/v) polyethylene glycol 8000 in citrate/phosphate buffer (pH 5.7) containing 0.5% (w/v) n-octyl-beta-D-glucopyranoside. The crystals reached maximum dimensions of 0.3 mm x 0.2 mm x greater than 2 mm. The enzyme crystallized in space group C222(1) with unit cell dimensions a = 246 A, b = 193 A and c = 75 A. A calculated Vm value of 2.35 A3/dalton was obtained assuming two molecules per asymmetric unit. The density of the crystals was found to be 1.16 g/ml, which confirmed the presence of two molecules per asymmetric unit and indicated a solvent content of 47.5%.

Lipoxygenase isoenzymes: a spectroscopic and structural characterization of soybean seed enzymes.

Applying recent developments in protein purification techniques, a number of lipoxygenase isoenzymes have been isolated in satisfactory quantities for a detailed physical and structural characterization. Four seed isoenzymes from two soybean cultivars have been studied by peptide mapping, free thiol and iron content determinations, and C-terminal analysis as well as by uv-visible absorption and EPR spectroscopy. While differences between the type 1 enzyme and the other isoenzymes were readily detected using proteolytic peptide mapping, digestion with dilute hydrochloric acid and C-terminal analysis both revealed structural features which were similar in all of the isoenzymes. One clear difference between the lipoxygenases was in their free sulfhydryl group content. The uv-visible absorption spectrum of each native isoenzyme was consistent with expectations for the experimental aromatic amino acid content. All of the isoenzymes contained one non-heme iron atom per molecule of protein. The oxidation of each isoenzyme with product hydroperoxide resulted in the conversion of the iron from an EPR silent state into several forms with EPR signals characteristic of high spin iron(III). The EPR spectra of all isoenzymes were remarkably similar. A time course EPR and catalytic activity study revealed that the various EPR active states represent a complex equilibrium between iron atoms in different environments. The pH dependence for the EPR and absorption spectroscopy lends support to the hypothesis that acid/base chemistry represents an important aspect of lipoxygenase catalysis.

Oxygenation of trans polyunsaturated fatty acids by lipoxygenase reveals steric features of the catalytic mechanism.

Lipoxygenase, a nonheme iron dioxygenase, catalyzes the oxygenation of 1,4-diene units in polyunsaturated fatty acids, forming conjugated diene hydroperoxides as the primary products. The naturally occurring all-Z geometry for the olefins in the polyunsaturated fatty acid has long been thought to be a substrate requirement for the enzyme. A rigorous test of this hypothesis using the two isomeric (9E,12Z)- and (9Z,12E)-9,12-octadecadienoic acids was carried out. Both isomeric substrates were found to be catalytically oxygenated by soybean lipoxygenase 1 at a significant fraction of the rate of the reaction of the natural substrate, linoleic acid. Product determinations revealed that a thermodynamically unfavorable E to Z isomerization at the 9,10-position occurred when (9E,12Z)-9,12-octadecadienoic acid was converted into the 13-hydroperoxide by lipoxygenase 1. Determination of the stereochemistry at the oxygenated position in the products indicated that a comparable isomerization at the 12,13-position did not occur when the 9Z,12E isomer was employed. The distribution of products obtained from oxygenation at the 9-position supported the hypothesis that the enzyme catalyzes the reaction in one of two substrate orientations, conventional and head to tail reversed. The observations can be understood on the basis of the steric demands on intermediates in the proposed mechanism of action as well as by catalysis by the active-site iron atom.

The electroanalytical approach to lipid peroxide determinations.

An electroanalytical method for the determination of lipid peroxides in physiological material is described. The technique is based on electrochemical detection for HPLC as the means for enhancing sensitivity. Samples containing organic peroxides, including lipid peroxides, can be analyzed directly using a modified polarographic detector (Lloyd, J.B.F.; Optimization of the operational parameters of the supported mercury drop electrode detector in high performance liquid chromatography. Anal. Chim. Acta 154:121-131; 1983.) for reversed phase HPLC determinations. Detection limits for fatty acid hydroperoxides were found to be in the low nanogram range.

The lipoxygenases in developing soybean seeds, their characterization and synthesis in vitro.

A number of lipoxygenase isoenzymes were identified in developing soybean (Glycine max L. Merrill cv Provar) seeds and two have been partially characterized. In a study of lipoxygenase level in developing soybean seeds, the enzyme content increased markedly during development. Comparisons of the lipoxygenases from mature soybean seeds and immature seeds by isoelectric focusing, chromatofocusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping identified two categories of isoenzyme. The isoenzymes from immature seeds were found by electron paramagnetic resonance spectroscopy to be isolated at least in part as the high spin iron(III) or active form of the enzyme in contrast to lipoxygenases from mature seeds which were isolated as electron paramagnetic resonance silent, high spin iron(II) species. The discovery of increased levels of lipoxygenases during seed development and their isolation in an active form suggests that the enzyme may play a physiological role during the maturation process. The incorporation of iron-59 from the nutrient medium into lipoxygenase during culture of immature seeds was indicative of de novo synthesis of the enzyme. The efficiency of the iron uptake was high, as indicated by the level of radioactivity found in the enzyme (one gram atom of iron per mole of lipoxygenase).

Determination of stereochemistry in the fatty acid hydroperoxide products of lipoxygenase catalysis.

High-performance liquid chromatography has been found to be an effective method for the determination of absolute configuration in the products of the lipoxygenase-catalyzed oxygenation of polyunsaturated fatty acids. Methyl esters of fatty acid hydroperoxides that had been reduced to the corresponding alcohols were converted into (+)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid esters. Enantiomeric alcohols were converted into diastereomeric esters that were readily resolved by normal-phase HPLC. The instrumental requirements for the technique are an isocratic HPLC and a uv absorbance monitor. The method was found to be effective in the determination of stereochemistry in the products derived from the action of plant lipoxygenases on linoleic acid. In addition, the chromatography of the derivatives obtained from lipoxygenase catalysis on arachidonic acid was found to be effective for the assignment of stereochemistry in those products. A comparison of the chromatography of these derivatives with that for the corresponding menthyloxycarbonyl derivatives demonstrated the superiority of this approach for the resolution of the diastereomeric pairs. The technique was applied to the determination of stereochemistry in the products derived from soybean lipoxygenase isoenzymes under a variety of experimental conditions.

Two geometrical isomers of linoleic acid: improved total syntheses.

The total syntheses of 9(Z),12(E)- and 9(E), 12(Z)-octadecadienoic acids have been carried out. A useful intermediate in both syntheses, 8-bromo-octanoic acid, recently has become available from commercial sources. This compound has been used to expedite the preparation of these isomers. The remaining carbon atoms were derived from propargyl alcohol along with either 1-heptyne or acetylene and 1-bromopentane. Because the overall yield for each sequence was roughly 15% and there were no extraordinary reaction conditions in any of the synthetic steps, the compounds could be prepared readily in multiple gram quantities. The syntheses of the two compounds were supported by data from a variety of spectroscopic techniques.

Resolution of the isoenzymes of soybean lipoxygenase using isoelectric focusing and chromatofocusing.

Isoelectric focusing in thin-layer polyacrylamide gels has been applied to the analysis of the enzymes involved in the formation and destruction of peroxides in soybeans [Glycine max (L.)], lipoxygenases and peroxidases, respectively. As a result of differences in pH optima for catalytic activity, lipoxygenases were selectively detected by adjusting the pH employed for activity-specific staining. Type-1 lipoxygenase was revealed not only by staining based on the conversion of linoleic acid to hydroperoxide but also by two stains based on the reduction of the hydroperoxide. These methods were found to be suitable for the analysis and characterization of isoenzyme patterns in different soybean cultivars. A substantial difference in the distribution of lipoxygenases maximally active near pH 7 was observed for cultivars Provar and Vickery. A similar degree of separation of the isoenzymes was achieved on a larger scale using chromato-focusing in the pH range 7.4-5.0.

A new class of lipoxygenase inhibitor. Polyunsaturated fatty acids containing sulfur.

A series of unsaturated and polyunsaturated fatty acids with a sulfur atom substituting for a methylene unit of the chain has been prepared and characterized. The syntheses were accomplished by the Wittig coupling of the yield derived from the triphenyl-phosphonium salt of 9-bromononanoic acid with aldehydes containing sulfur. The newly formed double bond had predominately the natural Z geometry even when the starting aldehyde was conjugated with the sulfur atom. The sulfides 13-thia-9(Z)-octadecenoic acid (2), 13-thia-9(Z), 11(E)-octadecadienoic acid (5) and 13-thia-9(E), 11(E)-octadecadienoic acid (6) were readily converted into their sulfoxide derivatives by treatment with an equivalent amount of m-chlorperoxybenzoic acid. The structures of the novel compounds were confirmed by the application of ir, uv, 1H-nmr, 13C-nmr, and (as methyl esters) chemical ionization mass spectrometry. Two members of this new family of fatty acids (5 and 6) were found to inhibit the catalysis of the oxygenation of linoleic acid by soybean type-1 lipoxygenase. The analysis of the kinetic data for compound 5 indicated that the type of inhibition was reversible competitive with an inhibition constant of 30 microM.