Biological transformations of polyunsaturated fatty acids often lead to chemically unstable products, such as the prostaglandin endoperoxides and leukotriene A 4 epoxide of mammalian biology and the allene epoxides of plants. Here, we report on the enzymatic production of a fatty acid containing a highly strained bicyclic four-carbon ring, a moiety known previously only as a model compound for mechanistic studies in chemistry. Starting from linolenic acid (C18.33), a dual function protein from the cyanobacterium Anabaena PCC 7120 forms 9R-hydroperoxy-C18.33 in a lipoxygenase domain, then a catalase-related domain converts the 9R-hydroperoxide to two unstable allylic epoxides. We isolated and identified the major product as 9R,10R-epoxy-11trans-C18.1 containing a bicyclo[1.1.0]butyl ring on carbons 13-16, and the minor product as 9R,10R-epoxy-11trans,13trans,15cis-C18.3, an epoxide of the leukotriene A type. Synthesis of both epoxides can be understood by initial transformation of the hydroperoxide to an epoxy allylic carbocation. Rearrangement to an intermediate bicyclobutonium ion followed by deprotonation gives the bicyclobutane fatty acid. This enzymatic reaction has no parallel in aqueous or organic solvent, where ring-opened cyclopropanes, cyclobutanes, and homoallyl products are formed. Given the capability shown here for enzymatic formation of the highly strained and unstable bicyclobutane, our findings suggest that other transformations involving carbocation rearrangement, in both chemistry and biology, should be examined for the production of the high energy bicyclobutanes.catalase ͉ carbocation ͉ epoxide ͉ leukotriene ͉ bicyclobutonium ion

Objective Black individuals are at an increased risk of myocardial infarction and stroke, two vascular diseases with strong thrombotic components. Platelet activation is a key step in platelet clot formation leading to myocardial infarction and stroke and recent work supports a racial difference in platelet aggregation through the thrombin protease-activated receptors (PARs). The underlying mechanism for this racial difference, however has not been established. Determining where in the signaling cascade these racial differences emerge will aid in understanding why individuals of differing racial ancestry may possess an inherent difference in their responsiveness to anti-platelet therapies. Approach and Results Washed human platelets from black volunteers were hyper-aggregable in response to PAR4-mediated platelet stimulation compared to whites. Interestingly, the racial difference in PAR4-mediated platelet aggregation persisted in platelets treated ex vivo with aspirin and 2MeSAMP, suggesting that the racial difference is independent of secondary feedback. Further, stimulation of platelets from black donors with PAR4-activating peptide (PAR4-AP) showed a potentiated level of activation through the Gq pathway compared to platelets from white donors. Differences in signaling included increased Ca2+ mobilization, Rap1 activation, and integrin αIIbβ3 activation with no observed difference in platelet protein expression between the groups tested. Conclusions Our study is the first to demonstrate that the Gq pathway is differentially regulated by race following PAR4 stimulation in human platelets. Furthermore, the racial difference in PAR4-mediated platelet aggregation persisted in the presence of COX and P2Y12 dual inhibition, suggesting that current anti-platelet therapy may provide less protection to blacks than whites.

Biosynthesis of the leukotriene A (LTA) class of epoxide is a lipoxygenase-catalyzed transformation requiring a fatty acid hydroperoxide substrate containing at least three double bonds. Here, we report on biosynthesis of a dienoic analog of LTA epoxides via a different enzymatic mechanism. Beginning with homolytic cleavage of the hydroperoxide moiety, a catalase/peroxidase-related hemoprotein from Anabaena PCC 7120, which occurs in a fusion protein with a linoleic acid 9R-lipoxygenase, dehydrates 9R-hydroperoxylinoleate to a highly unstable epoxide. Using methods we developed for isolating extremely labile compounds, we prepared and purified the epoxide and characterized its structure as 9R,10R-epoxy-octadeca-11E,13E-dienoate. This epoxide hydrolyzes to stable 9,14-diols that were reported before in linoleate autoxidation The plethora of products formed through oxygenation of polyunsaturated fatty acids was extended recently through characterization of the activities of an enzyme from Anabaena PCC 7120 (1), a photosynthetic cyanobacterium which is also termed Nostoc sp. PCC 7120 (2). The enzyme in question is a plasmid-encoded fusion protein with a catalase-related hemoprotein on the N-terminal side and a lipoxygenase at the C-terminal end. The catalase-related domain is 39 kDa in size, directly connected to the lipoxygenase (49 kDa), giving a molecular mass of 88 kDa for the full-length fusion protein. The full-length fusion protein was successfully expressed in Escherichia coli, and although the cDNA encoding only the catalase-related domain did not express as active protein, the cDNA of the lipoxygenase (LOX) domain expressed very well by itself, allowing the dual catalytic activities of the fusion protein to be clearly distinguished. The LOX domain converts C18 fatty acids to the corresponding 9R-hydroperoxide (3, 4). This is an unusual LOX activity, forming products enantiomeric to the 9S-LOX of plants (5), and characterized earlier in Hydra vulgaris (6), in the marine alga Ulva conglobata (7), and as an activity of Gersemia fruticosa arachidonate 11R-LOX (8). We showed that the catalaserelated domain converts the 9R-hydroperoxide of a-linolenic acid to two unstable epoxides that we isolated and characterized (1). One is an allylic 9,10-epoxide with C13-C16 of the carbon chain cyclized into a [1.1.0]bicyclobutane, a unique structural motif in biology. A second product, an allylic epoxide of the leukotriene A (LTA) type, 9R,10R-octadeca-11E,13E,15Z-trienoic acid, is also formed from the same 9R-hydroperoxylinolenic acid substrate (1).

Neuregulin (NRG) is an EGF-related growth factor that binds to the tyrosine kinase receptors ErbB3 and ErbB4, thus inducing tissue development and muscle glucose utilization during contraction. Here, we analyzed whether NRG has systemic effects regulating glycemia in control and type 2 diabetic rats. To this end, recombinant NRG (rNRG) was injected into Zucker diabetic fatty (ZDF) rats and their respective lean littermates 15 min before a glucose tolerance test (GTT) was performed. rNRG enhanced glucose tolerance without promoting the activation of the insulin receptor (IR) or insulin receptor substrates (IRS) in muscle and liver. However, in control rats, rNRG induced the phosphorylation of protein kinase B (PKB) and glycogen synthase kinase-3 (GSK-3) in liver but not in muscle. In liver, rNRG increased ErbB3 tyrosine phosphorylation and its binding to phosphatidylinositol 3-kinase (PI3K), thus indicating that rNRG activates the ErbB3/PI3K/PKB signaling pathway. rNRG increased glycogen content in liver but not in muscle. rNRG also increased the content of fructose-2,6-bisphosphate (Fru-2,6-P2), an activator of hepatic glycolysis, and lactate in liver but not in muscle. Increases in lactate were abrogated by wortmannin, a PI3K inhibitor, in incubated hepatocytes. The liver of ZDF rats showed a reduced content of ErbB3 receptors, entailing a minor stimulation of the rNRG-induced PKB/GSK-3 cascade and resulting in unaltered hepatic glycogen content. Nonetheless, rNRG increased hepatic Fru-2,6-P2 and augmented lactate both in liver and in plasma of diabetic rats. As a whole, rNRG improved response to the GTT in both control and diabetic rats by enhancing hepatic glucose utilization.

The composition-function relationship of HDL particles and its effects on the mechanisms driving coronary heart disease (CHD) is poorly understood. We tested the hypothesis that the functionality of HDL particles is significantly influenced by their lipid composition. Using a novel 3D-separation method, we isolated five different-sized HDL subpopulations from CHD patients who had low preβ-1 functionality (low-F) (ABCA1-dependent cholesterol-efflux normalized for preβ-1 concentration) and controls who had either low-F or high preβ-1 functionality (high-F). Molecular numbers of apoA-I, apoA-II, and eight major lipid classes were determined in each subpopulation by LC-MS. The average number of lipid molecules decreased from 422 in the large spherical α-1 particles to 57 in the small discoid preβ-1 particles. With decreasing particle size, the relative concentration of free cholesterol (FC) decreased in α-mobility but not in preβ-1 particles. Preβ-1 particles contained more lipids than predicted; 30% of which were neutral lipids (cholesteryl ester and triglyceride), indicating that these particles were mainly remodeled from larger particles not newly synthesized. There were significant correlations between HDL-particle functionality and the concentrations of several lipids. Unexpectedly, the phospholipid:FC ratio was significantly correlated with large-HDL-particle functionality but not with preβ-1 functionality. There was significant positive correlation between particle functionality and total lipids in high-F controls, indicating that the lipid-binding capacity of apoA-I plays a major role in the cholesterol efflux capacity of HDL particles. Functionality and lipid composition of HDL particles are significantly correlated and probably both are influenced by the lipid-binding capacity of apoA-I.