Max O. Funk

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 Å 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 Cα rms deviation of 1 Å). 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 stereospecificity 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). Proteins 29:15–31, 1997.

Structure-based design of a thiazolidinedione which targets the mitochondrial protein mitoNEET

Several PPAR-gamma agonists containing a thiazolidinedione moiety (referred to as glitazones) have been proposed to be neuroprotective and appear to alter mitochondrial function. Recently, a search for mitochondrial proteins that bind pioglitazone identified a novel protein, mitoNEET, which was later shown to regulate the oxidative capacity of the mitochondria. This identified an alternative target for the glitazones suggesting a possible new drug target for the treatment of neurodegenerative diseases. Molecular docking studies employing the reported crystal structure revealed five possible binding pockets on mitoNEET. We focused on two sites based on their physical characteristics. Using binding information gained from the analysis of two glitazones docked in these pockets, we designed and synthesized a ligand (NL-1) that would preferentially bind to site 1. Based on [(3)H]-binding data of the glitazones and comparisons to computer generated K(i)s, we were able to predict that site 1 was likely the target of the glitazones. NL-1 uncoupled isolated mitochondrial complex I respiration with an IC(50) of 2.4 microM and inhibited state III respiration up to 45%. To investigate the ability of NL-1 to block rotenone initiated free radicals from complex I, we found it was able to protect the human neuronal cell line SH-SY5Y against rotenone induced cell death. These data demonstrate that mitoNEET is a viable target for the design and synthesis of novel therapeutic agents aimed at altering mitochondrial function.

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.

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.

Identification of novel monoamine oxidase B inhibitors by structure-based virtual screening

Parkinsons disease is a severe debilitating neurodegenerative disorder. Recently, it was shown that the peroxisome proliferating-activator receptor-gamma agonist pioglitazone protected mice from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity due to its ability to inhibit monoamine oxidase B (MAO-B). Docking studies were initiated to investigate pioglitazones interactions within the substrate cavity of MAO-B. Modeling studies indicated that the thiazolidinedione (TZD) moiety was a likely candidate for its specificity to MAO-B. To explore this potential novel MAO-B scaffold, we performed a structure-based virtual screen to identify additional MAO-B inhibitors. Our search identified eight novel compounds containing the TZD-moiety that allowed for a limited study to identify structural requirements for binding to MAO-B. Inhibition assays identified two TZDs (A6355 and L136662) which were found to inhibit recombinant human MAO-B with IC(50) values of 82 and 195 nM, respectively.

Factors affecting the initial rate of lipoxygenase catalysis

Abstract The rate of peroxidation of linoleic acid by soybean type-1 lipoxygenase was studied under conditions which assured that the substrate was present as a monomolecular solution and that the first 5% of the reaction was observed. In order to achieve this, the kinetics were carried out at pH 10.0 in borate buffer using linoleic acid and enzyme concentrations of less than 75 μM and 0.2 nM respectively. The initial rate was increased by the presence of added product (13-hydroperoxy-9(Z),11(E)-octadecadienoic acid) in the substrate solutions in a concentration dependent and saturatable fashion. Product analogues lacking the hydroperoxide group (13-hydroxy-9(Z),11(E)-octadecadienoic acid and 13-methoxy-9(Z),11(E)-octadecadienoic acid) did not evoke this rate enhancing effect. These compounds reduced the initial rate when preincubated with enzyme prior to mixing with substrate. The results indicated that the chemical reactivity of the product was a necessary requirement for its activating effect on the enzyme.

A scaffold hopping approach to identify novel monoamine oxidase B inhibitors.

Monoamine oxidase B (MAO-B) inhibitors are used to treat Parkinsons disease. In this study, we searched for novel MAO-B inhibitors using a scaffold hopping approach based on our experience with the thiazolidinedione (TZD) class of compounds as MAO-B inhibitors. Several novel compounds were identified, with potencies in the low nanomolar and low micromolar range. We also found that derivatives of the natural product sulfuretin are potent MAO-A and MAO-B inhibitors.

Structure-activity relationship and docking studies of thiazolidinedione-type compounds with monoamine oxidase B.

The neuroprotective activity of pioglitazone and rosiglitazone in the MPTP parkinsonian mouse prompted us to evaluate a set of thiazolidinedione (TZD) type compounds for monoamine oxidase A and B inhibition activity. These compounds were able to inhibit MAO-B over several log units of magnitude (82 nM to 600 μM). Initial structure-activity relationship studies identified key areas to modify the aromatic substituted TZD compounds. Primarily, substitutions on the aromatic group and the TZD nitrogen were key areas where activity was enhanced within this group of compounds.

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.

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.