Mechanism Of Desaturation Research Articles

Spectroscopic and computational studies of a bifunctional iron- and 2-oxoglutarate dependent enzyme, AsqJ.

Iron and 2-oxoglutarate dependent (Fe/2OG) enzymes exhibit an exceedingly broad reaction repertoire. The most prevalent reactivity is hydroxylation, but many other reactivities have also been discovered in recent years, including halogenation, desaturation, epoxidation, endoperoxidation, epimerization, and cyclization. To fully explore the reaction mechanisms that support such a diverse reactivities in Fe/2OG enzyme, it is necessary to utilize a multi-faceted research methodology, consisting of molecular probe design and synthesis, in vitro enzyme assay development, enzyme kinetics, spectroscopy, protein crystallography, and theoretical calculations. By using such a multi-faceted research approach, we have explored reaction mechanisms of desaturation and epoxidation catalyzed by a bi-functional Fe/2OG enzyme, AsqJ. Herein, we describe the experimental protocols and computational workflows used in our studies.

Cavitation‐Associated Water Evaporation From Porous Media Through In Situ NMR Characterization

AbstractEvaporation of water from porous media is essential for a large variety of applications, whereas the opacity of porous matrix imposes considerable challenges in unveiling complicated phase‐change phenomena. Air invasion was previously reported as the major desaturation mechanism, while cavitation in porous media is not well studied. Herein we characterize the transient distribution and evaporation of water in homogeneous tight porous media with nondestructive nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI). By monitoring the amplitude change of NMR transverse relaxation time T2, we investigate the dynamical pore filling status and water content during evaporation. Interestingly, we find that the T2 spectrum shifts immediately after the evaporation starts, indicating the emptying of big pores from the entire medium. Disconnected void clusters at different depths in the porous medium are also observed from MRI scanning and optical images. These observations indicate the emergence of cavitation across the entire porous media along with the evaporation from open surface. Cavitation occurs when the water is stretched to metastable state by large capillary pressure from the evaporating meniscus. By studying the evaporation from hydrophilic membrane‐separated porous media, we further demonstrate the existence of cavitation‐associated evaporation. The preferential water vaporization from the bottom part can still be found from T2 spectrum analysis and optical imaging even when the water‐permeable membrane cuts off possible air invasion. Our findings confirm cavitation‐associated evaporation is one of the primary mechanisms for tight porous media, which provides valuable guidance for evaporation and moisture control.

Radical Rebound Hydroxylation Versus H-Atom Transfer in Non-Heme Iron(III)-Hydroxo Complexes: Reactivity and Structural Differentiation.

The characterization of high-valent iron centers in enzymes has been aided by synthetic model systems that mimic their reactivity or structural and spectral features. For example, the cleavage of dioxygen often produces an iron(IV)-oxo that has been characterized in a number of enzymatic and synthetic systems. In non-heme 2-oxogluterate dependent (iron-2OG) enzymes, the ferryl species abstracts an H-atom from bound substrate to produce the proposed iron(III)-hydroxo and caged substrate radical. Most iron-2OG enzymes perform a radical rebound hydroxylation at the site of the H-atom abstraction (HAA); however, recent reports have shown that certain substrates can be desaturated through the loss of a second H atom at a site adjacent to a heteroatom (N or O) for most native desaturase substrates. One proposed mechanism for the removal of the second H-atom involves a polar-cleavage mechanism (electron transfer-proton transfer) by the iron(III)-hydroxo, as opposed to a second HAA. Herein we report the synthesis and characterization of a series of iron complexes with hydrogen bonding interactions between bound aquo or hydroxo ligands and the secondary coordination spherein ferrous and ferric complexes. Interconversion among the iron species is accomplished by stepwise proton or electron addition or subtraction, as well as H-atom transfer (HAT). The calculated bond dissociation free energies (BDFEs) of two ferric hydroxo complexes, differentiated by theirnoncovalent interactions and reactivity, suggest that neither complex is capable of activating even weak C-H bonds, lending further support to the proposed mechanism for desaturation in iron-2OG desaturase enzymes. Additionally, the ferric hydroxo species are differentiated by their reactivity toward performing a radical rebound hydroxylation of triphenylmethylradical. Our findings should encourage further study of the desaturase systems that may contain unique H-bonding motifs proximal to the active site that help bias substrate desaturation over hydroxylation.

Mechanisms of Cytochrome P450-Catalyzed Oxidations.

Enzymes are complex biological catalysts and are critical to life. Most oxidations of chemicals are catalyzed by cytochrome P450 (P450, CYP) enzymes, which generally utilize mixed-function oxidase stoichiometry, utilizing pyridine nucleotides as electron donors: NAD(P)H + O2 + R → NAD(P)+ + RO + H2O (where R is a carbon substrate and RO is an oxidized product). The catalysis of oxidations is largely understood in the context of the heme iron-oxygen complex generally referred to as Compound I, formally FeO3+, whose basis was in peroxidase chemistry. Many X-ray crystal structures of P450s are now available (≥ 822 structures from ≥146 different P450s) and have helped in understanding catalytic specificity. In addition to hydroxylations, P450s catalyze more complex oxidations, including C-C bond formation and cleavage. Enzymes derived from P450s by directed evolution can even catalyze more unusual reactions, e.g. cyclopropanation. Current P450 questions under investigation include the potential role of the intermediate Compound 0 (formally FeIII-O2 -) in catalysis of some reactions, the roles of high- and low-spin forms of Compound I, the mechanism of desaturation, the roles of open and closed structures of P450s in catalysis, the extent of processivity in multi-step oxidations, and the role of the accessory protein cytochrome b 5. More global questions include exactly how structure drives function, prediction of catalysis, and roles of multiple protein conformations.

Insights into the Desaturation of Cyclopeptin and its C3 Epimer Catalyzed by a non‐Heme Iron Enzyme: Structural Characterization and Mechanism Elucidation

AbstractAsqJ, an iron(II)‐ and 2‐oxoglutarate‐dependent enzyme found in viridicatin‐type alkaloid biosynthetic pathways, catalyzes sequential desaturation and epoxidation to produce cyclopenins. Crystal structures of AsqJ bound to cyclopeptin and its C3 epimer are reported. Meanwhile, a detailed mechanistic study was carried out to decipher the desaturation mechanism. These findings suggest that a pathway involving hydrogen atom abstraction at the C10 position of the substrate by a short‐lived FeIV‐oxo species and the subsequent formation of a carbocation or a hydroxylated intermediate is preferred during AsqJ‐catalyzed desaturation.

Insights into the Desaturation of Cyclopeptin and its C3 Epimer Catalyzed by a non-Heme Iron Enzyme: Structural Characterization and Mechanism Elucidation.

AsqJ, an iron(II)- and 2-oxoglutarate-dependent enzyme found in viridicatin-type alkaloid biosynthetic pathways, catalyzes sequential desaturation and epoxidation to produce cyclopenins. Crystal structures of AsqJ bound to cyclopeptin and its C3 epimer are reported. Meanwhile, a detailed mechanistic study was carried out to decipher the desaturation mechanism. These findings suggest that a pathway involving hydrogen atom abstraction at the C10 position of the substrate by a short-lived FeIV -oxo species and the subsequent formation of a carbocation or a hydroxylated intermediate is preferred during AsqJ-catalyzed desaturation.

Crystal structure of human stearoyl-coenzyme A desaturase in complex with substrate.

Stearoyl-coenzyme A desaturase-1 (SCD1) has an important role in lipid metabolism, and SCD1 inhibitors are potential therapeutic agents for the treatment of metabolic diseases and cancers. Here we report the 3.25-Å crystal structure of human SCD1 in complex with its substrate, stearoyl-coenzyme A, which defines the new SCD1 dimetal catalytic center and reveals the determinants of substrate binding to provide insights into the catalytic mechanism of desaturation of the stearoyl moiety. The structure also provides a mechanism for localization of SCD1 in the endoplasmic reticulum: human SCD1 folds around a tight hydrophobic core formed from four long α-helices that presumably function as an anchor spanning the endoplasmic reticulum membrane. Furthermore, our results provide a framework for the rational design of pharmacological inhibitors targeting the SCD1 enzyme.

Sleep disorders in COPD: the forgotten dimension

Sleep in chronic obstructive pulmonary disease (COPD) is commonly associated with oxygen desaturation, which may exceed the degree of desaturation during maximum exercise, both subjectively and objectively impairing sleep quality. The mechanisms of desaturation include hypoventilation and ventilation to perfusion mismatching. The consequences of this desaturation include cardiac arrhythmias, pulmonary hypertension and nocturnal death, especially during acute exacerbations. Coexistence of COPD and obstructive sleep apnoea (OSA), referred to as overlap syndrome, has been estimated to occur in 1% of the general adult population. Overlap patients have worse sleep-related hypoxaemia and hypercapnia than patients with COPD or OSA alone. OSA has a similar prevalence in COPD as in a general population of similar age, but oxygen desaturation during sleep is more pronounced when the two conditions coexist. Management of sleep-related problems in COPD should particularly focus on minimising sleep disturbance via measures to limit cough and dyspnoea; nocturnal oxygen therapy is not generally indicated for isolated nocturnal hypoxaemia. Treatment with continuous positive airway pressure alleviates hypoxaemia, reduces hospitalisation and pulmonary hypertension, and improves survival.

A Saturation Suppression Approach for the Current Transformer—Part II: Performance Evaluation

Part I of this two-part paper introduces a hardware-based mechanism to prevent/suppress the saturation phenomenon of the current transformer (CT). The mechanism includes an electronically switched resistor which is in series with the CT secondary winding. Part II performs a set of 1) offline digital time-domain simulation studies in the PSCAD/EMTDC environment and 2) control-hardware-in-the-loop (CHIL) test cases in a real-time digital simulation platform, to demonstrate technical feasibility of the proposed approach. The investigations also report the effect of CT saturation and the proposed desaturation mechanism on a digital distance relay. The digital algorithms of the relay and the control of the electronic switch are implemented in two NI-CRIO platforms for the CHIL studies. The investigation results demonstrate and verify the effectiveness of the proposed CT desaturation mechanism.

Role of Ferredoxin and Flavodoxins in Bacillus subtilis Fatty Acid Desaturation

The Bacillus subtilis acyl lipid desaturase (Δ5-Des) is an iron-dependent integral membrane protein able to selectively introduce double bonds into long-chain fatty acids. In the last decade since its discovery, the molecular mechanism of Δ5-Des expression has been studied extensively. However, the mechanism of desaturation, which must rely on unknown bacterial proteins for electron transfer, has not yet been explored. The B. subtilis genome encodes three proteins that can act as potential electron donors of Δ5-Des, ferredoxin (Fer) and two flavodoxins (Flds) (YkuN and YkuP), which are encoded by the ykuNOP operon. Here we report that the disruption of either the fer gene or the ykuNOP operon decreases the desaturation of palmitic acid by ∼30%. Nevertheless, a fer ykuNOP mutant abolished the desaturation reaction almost completely. Our results establish Fer and the two Flds as redox partners for Δ5-Des and suggest that the Fer and Fld proteins could function physiologically in the biosynthesis of unsaturated fatty acids in B. subtilis. Although Flds have extensively been described as partners in a number of redox processes, this is the first report describing their role as electron donors in the fatty acid desaturation reaction.

Characteristics of Exercise‐Induced Intrapulmonary Arteriovenous Fistula in Patients with Unexplained Exertional Dyspnea

Dynamic appearance of intrapulmonary arteriovenous fistula (AVF) during exercise may be associated with unexplained exertional dyspnea (UED) and can be diagnosed with an agitated saline contrast study during exercise echocardiography. However, the occurrence of AVF during exercise in patients with UED has not been well described. Thus, the frequency of exercise-induced intrapulmonary AVF in the outpatients with UED was retrospectively analyzed. Thirty-nine outpatients (age: 53 ± 12, 33 female) with UED underwent symptom-limited supine bicycle exercise echocardiography. Ten patients (26%) developed exercise-induced intrapulmonary AVF. Patients with and without AVF showed the similar peak exercise heart rate, systolic blood pressure, and rate-pressure product. The patients with AVF demonstrated a small but significant decrease in arterial oxygen saturation with exercise as compare to baseline (95.6 ± 2.8% at peak, vs. 97.5 ± 2.5% at baseline, P < 0.05 with a paired Student t-test). Our study suggests that exercise-induced intrapulmonary AVF is relatively common in the outpatients with UED and associated with mild exercise desaturation; however, the mechanism of desaturation could not be determined by this study. Further investigation to characterize and determine the clinical significance of AVF is warranted.

Occurrence and mechanisms of sudden oxygen desaturation in infants who sleep face down.

Infants who sleep prone and face down on soft bedding are particularly vulnerable for sudden infant death syndrome. It has been suggested that 1 mechanism of death in this situation involves rebreathing of expired air. Many infants tolerate rebreathing while lying prone face down for long periods with stable saturations. Others occasionally have rapid desaturations and may require intervention to terminate rebreathing. The present study had 3 objectives: 1) to determine the frequency of rapid desaturations in a large group of healthy infants, 2) to elucidate the mechanism of these desaturations, and 3) to determine the timing of these events during periods of rebreathing. We studied respiratory tracings and videotapes of 56 healthy 1- to 6-month-old infants who were sleeping face down and rebreathing on soft bedding in our laboratory. We compared the frequency of desaturations during rebreathing and nonrebreathing periods. We measured respiratory frequency and apnea occurrence before desaturation and nonrebreathing control episodes. We also measured minute ventilation during steady state before desaturation and just before desaturation. There were 25 desaturation episodes in infants while rebreathing, occurring in 11 (19.6%) of the 56 infants. Episodes were significantly more frequent during rebreathing than during nonrebreathing periods. Three desaturation episodes reached <85%; 2 required intervention to terminate rebreathing. The respiratory frequency was not different between nonrebreathing control and desaturation episodes. Brief apneas were significantly more frequent preceding desaturation than control episodes (44% vs 4%). Just before episodes, there was a transient decrease in minute volume despite increasing inspired carbon dioxide in 3 episodes. There was evidence of partial or complete pharyngeal airway obstruction in 3 episodes. Thirty-six percent of all episodes were immediately preceded by behavioral arousal. Rebreathing in prone sleeping infants is associated with an increased frequency of episodic desaturations. Desaturation may result from respiratory pattern changes such as brief apneas often associated with evidence of behavioral arousal or failure to increase ventilation in the face of rising inspired carbon dioxide, also associated with behavioral arousal.

Desaturation, chain scission, and register-shift of oxygen-substituted fatty acids during reaction with stearoyl-ACP desaturase.

Stearoyl acyl carrier protein Delta(9) desaturase catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C-9 and C-10 positions of the acyl chain in the kinetically preferred natural substrate 18:0-ACP. In this work, substrate analogues with an oxygen atom singly replacing the methylene groups at the 8, 9, 10, and 11 positions of the stearoyl chain were synthesized, converted to acyloxy-ACPs, and used as probes of desaturase reactivity. Evidence for desaturation, acyloxy chain scission, and register-shift in binding prior to chain scission was obtained. Reactions with acyloxy-ACPs having either O-8 or O-11 substitutions gave a single desaturation product consistent with the insertion of a cis double bond between C-9 and C-10. The k(cat)/K(M) values for the O-8- and O-11-substituted acyloxy-ACPs were comparable to that of the natural substrate, indicating that the presence of an ether group adjacent to the site of reactivity did not significantly interfere either with the desaturation reaction or with the binding of substrate in the proper register for desaturation between C-9 and C-10. For reactions with the O-9 and O-10 acyloxy-ACPs, the k(cat) values were decreased to approximately 3% of that observed for 18:0-ACP, and upon reaction, the acyloxy chain was broken to yield an omega-hydroxy fatty alkanoyl-ACP and a volatile long-chain aldehyde. For the O-9 substitution, 8-hydroxyoctanoate and 1-nonanal were obtained, corresponding to the anticipated binding register and subsequent reaction between the O-9 and C-10 positions. In contrast, the O-10 substitution yielded 9-hydroxynonanoyl-ACP and 1-octanal, corresponding to an obligate "register-shift" of acyloxy chain binding prior to reaction between the O-10 and C-11 positions. Register-shift is thus defined as a mechanistically relevant misalignment of acyl chain binding that results in reaction at positions other than between C-9 and C-10. The inability of the O-10 acyloxy probe to undergo reaction between the C-9 and O-10 positions provides evidence that the Delta9D-catalyzed desaturation of stearoyl-ACP may initiate at C-10. Possible mechanisms of the acyl chain scission and implications of these results for the desaturation mechanism are considered.

Development of an attitude control system for a balloon-borne gamma ray telescope

Abstract We describe the attitude control system employed by the MASCO balloon-borne gamma ray telescope and present the first laboratory tests of this system. The MASCO experiment is a low energy gamma ray imaging telescope that employs a MURA coded mask. Its angular resolution is 14′ over a 13° field of view, which requires a pointing accuracy of some arcminutes in order to allow the accomplishment of the scientific goals. The attitude control system was designed to provide pointing and stabilization of a 1400 kg, 7 m-high, 2 m-wide, 2 m-deep gondola that encompasses the telescope detector system and its associated electronics. The sensors used are an electronic magnetic compass, an encoder, a two-axis solar sensor, a sun-tracker, two accelerometers, a gyroscope and three CCD stellar sensors. A GPS system is also employed. The actuators are a reaction wheel for azimuth control, an azimuth motor located in the balloon-gondola decoupling mechanism for desaturation of the reaction wheel, and a telescope elevation motor with a harmonic drive reduction. The software architecture and the operation modes are also presented.

Control of unsaturated fatty acid substituents in emulsans

The ability to regulate the content of unsaturated fatty acids (FAs) of emulsans (EMs) formed by Acinetobacter calcoaceticus RAG-1 was studied. Studies of EM biosynthesis with 13C 1-labeled FAs demonstrated that 95±7% of 16:1(9- cis) incorporated into EMs (EM-FAs) were formed by desaturation of the carbon source 16:0. An aerobic desaturation mechanism involving Δ-9 desaturase activity was proposed to explain these results. The direct incorporation of Δ-9- cis unsaturated acids occured concurrently with a decrease in the content of other 9- cis unsaturated EM-FAs. Important factors which ultimately determined the composition of unsaturated EM-FAs were the following: (i) feedback inhibition of Δ-9 desaturase activity, (ii) direct incorporation of FAs from a carbon source and (iii) two-carbon unit elongation or removal. The incorporation of polyunsaturated FAs into EMs was also accomplished by the selective feeding method. For example, by feeding RAG-1 with 18:2(9,12- trans), an EM was formed that contained almost 55 nmol/mg-EM (GC-MS). The surface activities of the new EMs from unsaturated FAs were evaluated.

Plasma phospholipid fatty acids in X-linked adrenoleukodystrophy

Blood cell and plasma lipid classes and their fatty acids were analyzed in a child with X-linked adrenoleukodystrophy. The increase in saturated fatty acids with very long chains typical of this disease occurred almost exclusively in sphingomyelin. In this lipid, the proportion of lignoceric (24:0) and hexacosanoic (26:0) acids increased while that of 18:0, 20:0, and 24:1 decreased. In the rest of the lipid classes, but especially in cholesteryl esters and triacylglycerols, the proportion of linoleate (18:2) decreased while that of oleate (18:1) increased. In glycerophospholipids, polyunsaturated fatty acids such as 20:4n-6, 22:5n-6, and 22:6n-3 were reduced while their immediate precursors, 20:3n-6, 22:4n-6, and 22:5n-3, respectively, were relatively increased, suggesting a defect in fatty acid desaturation mechanisms. Although less pronounced, a similar trend of changes was seen in the patient's mother; in both, all alterations were more marked in serum than in blood cells.

Distribution of Anacardic Acids Associated with Small Pest Resistance among Cultivars of Pelargonium ×hortorum

Composition of anacardic acids (phenolic acids known to be associated with small pest resistance in Pelargonium ×hortorum) was examined in 13 diploid and 25 tetraploid cultivars by high-performance liquid chromatography (HPLC). The presence of an unusual desaturation (omega (ω)-5) in the alkyl tail of anacardic acids present only in glandular trichome exudate of pest-resistant diploid inbred lines had previously been associated with a sticky-trap pest-resistance phenomenon. In this study, we examine Pelargonium cultivars for variability in anacardic acid composition to assess the distribution of ω5 desaturation among commercial cultivars, to determine possible interactions between ω5 desaturation and other plant desaturation mechanisms, and to examine the possible impact of ploidy on ω5 desaturation. An unsaturation index (UI) is derived to compare exudates differing widely in composition yet which may provide a similarly effective sticky-trap pest-resistance mechanism based on exudate viscosity. ω-5 Anacardic acids were observed in the glandular trichome exudate of all 38 commercial cultivars examined. No diploid cultivar produced ω5- and ω9- anacardic acids, although the simultaneous production of ω5 and ω9- anacardic acids was observed in three tetraploid cultivars. Total ω5- anacardic acids comprised from 42.4% (tetraploid cultivar Perlenkette-syn. Snowhite, Weiss) to 86.8% (tetraploid cultivar Amanda). Commercial P. ×domesticum cultivars had no ω5 anacardic acids. UIs ranged from 60.9 (tetraploid cultivar Dixieland) to 103.4 (diploid cultivar Pinto White). In contrast, anacardic acids collected from a pest-susceptible inbred line contained no ω5- anacardic acids and had a UI of 38.7. No significant differences among ploidy levels were observed for UIs or for most specific anacardic acid components, with the exception of 24:1 ω5- anacardic acid, in which the mean diploid value (32.1%) was significantly higher than that of the mean tetraploid value (27.6%). We conclude that ω5- anacardic acid production occurs in all Pelargonium cultivars observed and that these cultivars are predicted to exhibit resistance to small arthropod pests. Significant genetic variability in specific anacardic acid composition appears to exist among Pelargonium cultivars, suggesting that breeding for pest resistance can be readily monitored by HPLC of anacardic acids.

Inhibition and substrate specificity of yeast Δ 22-desanturase

Using yeast microsomes, 23-hydroxysterols were tested as intermediates in the formation of the sterol side delta 22-double bond. No evidence could be found supporting a two-stage mechanism of desaturation via hydroxylation and dehydration. Sterols with various side chains were tested as substrates. Those with alkyl substituents in the 24-alpha position were poor substrates. A series of sterols, including cyclopropyl sterols, were tested as mechanism-based inhibitors without success. Inhibition was observed with an isocyano-sterol.

Lipid-Linked Desaturation of Palmitic Acid in Monogalactosyl Diacylglycerol in the Blue-Green Alga (Cyanobacterium) Anabaena variabilis Studied in Vivo

The mechanism of desaturation of palmitic acid in monogalactosyl diacylglycerol in Anabaena variabilis was studied by labeling in vivo with 13C and mass spectrometry. When the cells were fed with [13C]Na2CO3 for 2.5 h, 19% of the palmitic, but virtually none of the palmitoleic, acid at the C-2 position of the lipid was enriched with 13C. During subsequent incubation for 7.5 h, the [13C]palmitic acid was desaturated to [13C]palmitoleic acid. Mass spectrometric analysis of the 2-acylglycerol moiety of the lipid indicated that [13C]palmitoyl-[13C]glycerol and [12C]palmitoyl-[12C]glycerol were converted to [13C]palmitoleoyl-[13C]glycerol and [12C]palmitoleoyl-[12C]glycerol, respectively. These results suggest that the palmitic acid was converted to palmitoleic acid in vivo by lipid-linked desaturation but not via a pathway consisting of deacylation, desaturation and reacylation.

A Complex Glycolipid as Possible Precursor in the Light Driven Biosynthesis of Monogalactosyl Diglyceride

Summary Short term 14C-incorporation kinetics of the lipid fraction of spinach leaf sections revealed a complex glycolipid, termed GL. The lipid is slightly more polar than monogalactosyl diglyceride (MGDG) and contains sugar, glycerol and fatty acids in a molar ratio of 1: 1: 1-2. The fatty acid pattern is similar to that of monogalactosyl diglyceride and the sugar moiety shows variable amounts of glucose and galactose dependent on the physiological state of the material. An increase in the galactose/glucose-ratio of this lipid is accompanied by an increasing degree of desaturation of its fatty acid pattern. The data are discussed in terms of a fatty acid desaturation mechanism outside the chloroplast, in which GL is supposed to have an acyl carrier function in the synthesis of monogalactosyl diglyceride.