Crosslinking Studies To Probe Substrate Binding By Soybean Lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids into conjugated diene hydroperoxides. The three dimensional structure of SBLO-1 is known, but it is not certain how substrates bind. One hypothesis involves the transient separation of helix-2 and helix-11 located on the exterior of the molecule in front of the active site iron. A second hypothesis involves a conformational change in the side chains of residues leucine 541 and threonine 259. To test these hypotheses, site directed mutagenesis was used to create a cysteine mutation on each helix, which could allow for the formation of a disulfide linkage. Disulfide formation between the two cysteines in the T259C,S545C mutant was found to be unfavorable, but later shown to be present at higher pH values using SDS-PAGE. Treatment of the T259C,S545C with the crosslinker 2,3-dibromomaleimide (DBM) resulted in a 50% reduction in catalytic activity. No loss of activity was observed when the single mutant, S545C, or the wild type was treated with DBM. Single mutants T259C and L541C both showed approximately 20% reduction in the rate after addition of DBM. Double mutants T259C,L541C and S263C,S545C showed approximately 30% reduction in the rate after addition of DBM. Single mutants T259C and L541C showed an increase in activity after incubation with NEM. Double mutants T259C,S545C and T259C,L541C showed an increase in activity after incubation with NEM. The S263C,S545C double mutant showed a slight decrease in activity in the presence of NEM. It is unclear how the NEM and DBM are interacting with the molecule, but this can easily be determined through mass spectrometry experiments.

Enzymatic Activity of Soybean Lipoxygenase-1 on Linoleoyl-Derivatized Agarose

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of linoleic acid to form 13(S) and 9(R) hydroperoxides. The manner in which substrates bind to the lipoxygenase family of enzymes is not known. It is believed fatty acid substrates may bind either with the aliphatic end first or with the carboxylate group facing the interior of the protein. This thesis tested a potential methyl-end first substrate binding mechanism by studying the activity of SBLO-1 to oxygenate immobilized linoleoyl residues attached to an insoluble polymer. Linoleic acid was attached to aminohexyl agarose in the presence of N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS). The concentration of the covalently attached residues was facilitated by enriching linoleic acid with a small amount of the radioactive 14C-isotope. Functionalization yields of 3% available primary amines on the resin were obtained. Enzymatic oxygenation of the linoleoyl-residues was verified using the ferrous oxidation in xylenol orange (FOX) assay. Approximately 30% of the attached linoleoyl moieties were converted to hydroperoxides in the presence of SBLO-1. A disulfide-containing cleavable linker, cystamine, was used as part of an improved method to isolate the product in a facile manner. Cystamine was attached to NHS-activated agarose with approximately 5% overall functionalization yield of available functional groups. 14C-linoleic acid was successfully covalently linked to the cystamine moieties in the presence of EDC and NHS. The FOX assay verified the enzymatic oxygenation of the linoleoyl residues attached to cystamine-derivatized agarose. The isolation of the peroxide product was attempted in a series of extractions in organic solvents. The product was analyzed using GC/MS which did not show a new peak indicative of product. Further work is needed to successfully analyze the stereoand regiochemistry of the oxygenated product. The presence of the peroxides in this study indicated the linoleoyl residues behave as substrates of SBLO-1. It is unknown how bulky substrates bind to the active site; however, it is difficult to rationalize a carboxylate group-first binding mode. Discovery of the 13(S)-hydroperoxide product on the linoleoyl-agarose would support the claim of a potential methyl-end first binding mechanism.

Study Of The Binding Of Substrate By The Phe557Val Mutant Soybean Lipoxygenase-1

Lipoxygenases are a class of enzymes which consist of non-heme iron dioxygenases that are produced by fungi, plants, and mammals and catalyze the oxygenation of polyunsaturated fatty acid substrates to unsaturated fatty acid hydroperoxide products. The unsaturated fatty acid hydroperoxide products are stereo- and regiospecific. One such lipoxygenase, soybean lipoxygenase-1 (SBLO-1), catalyzes the conversion of linoleate to 13-hydroperoxy-9(Z),11(E)-octadecadienoate (13-HPOD) and a small amount of 9-hydroperoxy-10(E),12(Z)-octadecadienoate (9-HPOD). Although the structure of SBLO-1 is known and it is the most widely studied lipoxygenase, how it binds to substrate is still poorly understood. Two competing binding hypotheses that have been used to understand and explain the binding are the head first binding model and the tail first binding model. The head first binding model predicts linoleate binds with its polar carboxylate group in the binding pocket and the methyl terminus at the surface of the binding pocket. The tail first binding model predicts that linoleate binds with its methyl terminus end in the binding pocket and the polar carboxylate group at the surface of the binding pocket. Both binding models have been used in the explanation of previous work. In previous work the replacement of phenylalanine with valine has been performed to produce the phe557val mutant SBLO-1. The mutant SBLO-1 was then used in the enzymatic oxygenation of linoleate. With this mutant, the amount of 9-HPOD that is formed increases. This result has been interpreted using the head-first binding model in which the smaller valine residue allows linoleate to bind with the polar carboxylate group of linoleate interacting with arginine-707. The work presented in this thesis confirms the regiochemical results of the previous work and further tests the head-first binding model. If head-first binding occurs, the 9-HPOD is expected to have primarily S configuration. Utilizing chiral-phase HPLC, it was found that the 9-HPOD produced by the phe557val mutant SBLO-1 is primarily S, consistent with head-first binding. The head-first binding model was also tested using linoleyl dimethylamine (LDMA), which has been shown to be a good substrate for SBLO-1 at pH 7.0, where LDMA is thought to be positively charged. This model predicts that less of the 9-peroxide should be produced with this substrate. Through the use of gas chromatography/mass spectrometry, it was found that the conversion of LDMA by the phe557val mutant SBLO-1 resulted in the formation of a 46:54 mixture of the 13-peroxide:9-peroxide. The higher amount of 9-peroxide is the opposite of what is expected for the currently proposed model suggesting that the proposed model may not be entirely correct. The results thus far have been consistent with reverse binding but not with the proposed interaction of the polar end of the substrate with arginine-707.

Copper and human health: biochemistry, genetics, and strategies for modeling dose-response relationships

Copper (Cu) and its alloys are used extensively in domestic and industrial applications. Cu is also an essential element in mammalian nutrition. Since both copper deficiency and copper excess produce adverse health effects, the dose-response curve is U-shaped, although the precise form has not yet been well characterized. Many animal and human studies were conducted on copper to provide a rich database from which data suitable for modeling the dose-response relationship for copper may be extracted. Possible dose-response modeling strategies are considered in this review, including those based on the benchmark dose and categorical regression. The usefulness of biologically based dose-response modeling techniques in understanding copper toxicity was difficult to assess at this time since the mechanisms underlying copper-induced toxicity have yet to be fully elucidated. A dose-response modeling strategy for copper toxicity was proposed associated with both deficiency and excess. This modeling strategy was applied to multiple studies of copper-induced toxicity, standardized with respect to severity of adverse health outcomes and selected on the basis of criteria reflecting the quality and relevance of individual studies. The use of a comprehensive database on copper-induced toxicity is essential for dose-response modeling since there is insufficient information in any single study to adequately characterize copper dose-response relationships. The dose-response modeling strategy envisioned here is designed to determine whether the existing toxicity data for copper excess or deficiency may be effectively utilized in defining the limits of the homeostatic range in humans and other species. By considering alternative techniques for determining a point of departure and low-dose extrapolation (including categorical regression, the benchmark dose, and identification of observed no-effect levels) this strategy will identify which techniques are most suitable for this purpose. This analysis also serves to identify areas in which additional data are needed to better define the characteristics of dose-response relationships for copper-induced toxicity in relation to excess or deficiency.

Large deletions in the CFTR gene: clinics and genetics in Swiss patients with CF

Cystic fibrosis (CF) is the most common life-shortening autosomal recessive disorder in Caucasians, and is associated with at least one mutation on each CF transmembrane conductance regulator (CFTR) allele. Some patients, however, with only one identifiable point mutation carry on the other allele, a large deletion that is not detected by conventional screening methods. The overall frequency of large deletions in patients with CF is estimated to be 1-3%. Using the CFTR Multiplex Ligation dependent Probe Amplification Kit (MRC-Holland, Amsterdam, Netherlands) that allows the exact detection of copy numbers from all 27 exons in the CFTR gene, we screened 50 patients with only one identified mutation for large deletions in the CFTR gene. Each detected deletion was confirmed using our real-time polymerase chain reaction (PCR) assay and deletion-specific PCR reactions using junction fragment primers. We detected large deletions in eight patients (16%). These eight CF alleles belong to four different deletion types (CFTRindel2, CFTRdele14b-17b, CFTRdele17a-17b and CFTRdele 2-9) whereof the last is novel. Comparing detailed clinical data of all these patients with CF and the molecular genetic findings, we were able to elaborate criteria for deletion screenings and possible genotype-phenotype associations. In conclusion, we agree with other authors that deletion screenings should be implemented in routine genetic diagnostics of CF.

Genetics of growth hormone deficiency

When a child is not following the normal, predicted growth curve, an evaluation for underlying illness and central nervous system abnormalities is required and appropriate consideration should be given to genetic defects causing growth hormone (GH) deficiency. This article focuses on the GH gene, the various gene alterations, and their possible impact on the pituitary gland. Transcription factors regulating pituitary gland development may cause multiple pituitary hormone deficiency but may present initially as GH deficiency. The role of two most important transcription factors, POU1F1 (Pit-1) and PROP 1, is discussed.

Sitosterolaemia in Switzerland: molecular genetics links the US Amish-Mennonites to their European roots

Sitosterolaemia is a rare autosomal recessive disease characterized by increased intestinal absorption of plant sterols, decreased hepatic excretion into bile and elevated concentrations in plasma phytosterols. Homozygous or compound heterozygous loss of function mutations in either of the ATP-binding cassette (ABC) proteins ABCG5 and ABCG8 explain the increased absorption of plant sterols. Here we report a Swiss index patient with sitosterolaemia, who presented with the classical symptoms of xanthomas, but also had mitral and aortic valvular heart disease. Her management over the last 20 years included a novel therapeutic approach of high-dose cholesterol feeding that was semi-effective. Mutational and extended haplotype analyses showed that our patient shared this haplotype with that of the Amish-Mennonite sitosterolaemia patients, indicating they are related ancestrally.