Flavour biogenesis. Partial purification and properties of a fatty acid hydroperoxide cleaving enzyme from fruits of cucumber

A membrane-bound enzyme, which catalyses the cleavage of fatty acid hydroperoxides to carbonyl fragments, has been partially purified from cucumber fruit. The isomeric 9- and 13-hydroperoxydienes (but not the hydroxydienes) derived from both linoleic and linolenic acids are cleaved by the enzyme but a mixture of 9- and 10-hydroperoxymonoenoic derivatives of oleic acid was not attacked. No evidence was obtained for free intermediates between fatty acid hydroperoxides and the cleavage products. Major volatile products were: cis-3-nonenal and hexanal (from 9- and 13-hydroperoxides of linoleic acid respectively) or cis-3,cis-6-nonadienal and cis-3-hexenal (from 9- and 13-hydroperoxides of linolenic acid). The increase in the ratio of cis-3- to trans-2-enal products with enzyme purification indicated that cis-3-enals are the immediate cleavage products and that the trans-2- forms are produced by subsequent isomerization.     

Biosynthetic pathway of cucumber alcohol: Trans-2,cis-6-nonadienol via cis-3,cis-6-nonadienal

cis-3,cis-6-Nonadienal and cis-3-nonenal in Cucumis sativus were identified by comparison with synthetic specimens. The identification of these compounds, combined with biochemical evidence, suggests that cucumber alcohol and trans-2-nonenol are biosynthesized via cis-3-unsaturated aldehydes from linolenic and linoleic acid, respectively.     

The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

Background

Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.

Results

Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding.

Conclusions

Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP³⁵RIW motif of N-terminal Vpr.     

Cyclisation of 1-trans-1′-norsqualene-2,3-epoxide and 1-cis-1′-norsqualene-2,3-epoxide by a cell free system of corn embryos

Squalene-2,3-epoxide-cycloartenol cyclase and cycloeucalenol-obtusifoliol isomerase activities were found in microsomal fractions of corn (Zea mays) embryos. Squalene-2,3-epoxide, 1-trans-1′-norsqualene-2,3-epoxide and 1-cis-1′-norsqualene-2,3-epoxide were incubated. Squalene-2,3-epoxide was cyclized giving only cycloartenol, whereas 1-trans-1′-norsqualene-2,3-epoxide gave 31-norcycloartenol and 31-norlanosterol with a reduced yield, 1-cis-1′-norsqualene-2,3-epoxide was not significantly cyclized.     

Preparation of methyl cis-9,cis-12-octadecadienoate-16,16,17,17-d4

An eight-step synthesis is described which gives an overall yield of ～30% methyl cis-9,cis-12-octadecadienoate-16,16,17,17-d₄. The preparation utilizes easily obtainable starting materials. Tris(triphenylphosphine)chlororhodium (I) catalyst is used for incorporation of the deuterium isotopes. The double bond in the 9 position is created by the Wittig coupling of 1-non-3-enyl-d₄-triphenylphosphonium bromide to methyl 8-formyloctanoate. Various methods for preparation of the intermediate and final products are discussed. Partial argentation resin chromatography was used to remove the ～9% trans/cis, cis/trans, and trans/trans isomers also produced. Analysis of the final product by mass spectrometry (MS) indicated 96%-d₄.     

Cis-4-hydroxypipecolic acid and 2,4-cis-4,5-trans-4,5-dihydroxypipecolic acid from Calliandra

cis-4-Hydroxypipecolic acid and 2,4-cis-4,5-trans-4,5-dihydroxypipecolic acid were isolated from leaves of Calliandra pittieri. A system for resolving the eight imino acids isolated from Calliandra is described.     

Biosynthesis of trans-2-hexenal in chloroplasts from Thea sinensis

The biosynthetic pathway of trans-2-hexenal, leaf aldehyde, in isolated chloroplasts of Thea sinensis leaves. was examined using a tracer experiment. A high and specific incorporation of radioactivity into cis-3-hexenal and trans-2-hexenal, was observed when linolenic acid-[U-¹⁴C] was incubated with the isolated chloroplasts. Thus, trans-2-hexenal was biosynthesized via cis-3-hexenal from linolenic acid in the chloroplasts.     

Studies on fluorescence polarization of 1-acyl-2-cis- or trans-parinaroyl sn-3-glycerophosphorylcholines in model systems and microsomal membranes

Fluorescent lecithin probes containing cis- or trans-parinaric acid (PnA) at the 2-position cis-parinaroylphosphatidylcholine (cis-PnPC) and trans-parinaroyl phosphatidylcholine (trans-PnPC)) showed similar behavior to that of the free cis- or trans-parinaric acids (cis-PnA or trans-PnA) in bilayer vesicles of synthetic saturated lecithins. Transition temperatures detected by cis-PnPc were about 1°C lower than those observed with trans-PnPc. In mixed lecithin vesicles, the trans-PnPc probe monitored a higher temperature melting component than did the cis-probe. Both probes were readily incorporated into microsomal membranes and into sonicated vesicles prepared from the microsomal phospholipids. With either cis- or trans-PnPc no change in polarization ratio was observed for microsomal membranes between 40°C and 0°C but this ratio increased with decreasing temperature between 0°C and ?5°C. However, vesicles of extracted phospholipids showed a continuous increase in polarization ratio with decreasing temperature between 20°C and ?15°C with trans-PnPc and bewteen 5°C and ?15°C with cis-PnPc. These results suggest that the two lecithin probes monitor different environments in the membranes and phospholipid vesicles prepared from them.     

Characterization of novel 2-oxoglutarate dependent dioxygenases converting l-proline to cis-4-hydroxy-l-proline

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing l-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-l-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-l-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel l-proline cis-4-hydroxylases converting free l-proline to cis-4-hydroxy-l-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected l-proline cis-4-hydroxylase activity in both proteins. Likewise l-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).     

Solvent exchange, solvent interchange, aquation and isomerisation reactions of cis- and trans-[Co(tmen)2(NCMe)2] in water, Me2SO and MeCN: kinetics and stereochemistry

The synthesis and characterisation of cis- and trans-[Co(tmen)₂(NCCH₃)₂](ClO₄)₃ are described. Solvolysis rates have been measured by both ¹H NMR spectroscopy and UV-Vis spectrophotometry in dimethyl sulfoxide at 298.2 K. The cis isomer undergoes solvolysis by consecutive first-order reactions, k₁=5.61 × 10⁻⁴ and k₂=5.35 × 10⁻⁴ s⁻¹, each with steric retention. The measured solvolysis rate (single step reaction) for the trans isomer is k=1.54 × 10⁻⁵ s⁻¹. The solvent exchange rates have been measured by ¹H NMR spectroscopy in CD₃CN at 298.2 K: k_ex(cis)=k_ct + k_cc=2.0 × 10⁻⁵ and k_ex(trans)=k_tc + k_tt=4.56 × 10⁻⁶ s⁻¹. From these data, the measured cis-trans isomerisation rate (1.71 × 10⁻⁶ s⁻¹) and equilibrium position in CH₃CN (17% trans), the steric course for substitution in the exchange processes has been determined: trans reactant - 69% trans product; cis reactant - 99% cis product. Aquation rates for cis- and trans-[Co(tmen)₂(NCCH₃)₂](ClO₄)₃ have also been determined spectrophotometrically and by NMR; k_cis=1.3 × 10⁻⁴ and k_trans=2.7 × 10⁻⁵ s⁻¹. In both cases the steric course for the primary aquation step is indeterminate because the subsequent steps are faster. Where data are available, the [Co(tmen)₂X₂]ⁿ⁺ complexes are found to be consistently much more reactive than their [Co(en)₂X₂]ⁿ⁺ analogues.     

Different reaction mechanisms for cis- and trans-prenyltransferases

Octaprenyl diphosphate synthase (OPPs) and undecaprenyl diphosphate synthases (UPPs) catalyze consecutive condensation reactions of farnesyl diphosphate (FPP) with 5 and 8 isopentenyl diphosphate (IPP) to generate C₄₀ and C₅₅ products with trans- and cis-double bonds, respectively. In this study, we used IPP analogue, 3-bromo-3-butenyl diphosphate (Br-IPP), in conjunction with radiolabeled FPP, to probe the reaction mechanisms of the two prenyltransferases. Using this alternative substrate with electron-withdrawing bromo group at the C3 position to slow down the condensation step, trapping of farnesol in the OPPs reaction from radiolabeled FPP under basic condition was observed, consistent with a sequential mechanism. In contrast, UPPs reaction yielded no farnesyl carbocation intermediate under the same condition with radiolabeled FPP and Br-IPP, indicating a concerted mechanism. Our data demonstrate the different reaction mechanisms for cis- and tran-prenyltransferases although they share the same substrates.     

Partial purification and characterization of the linoleate hydroperoxide isomerase from grains of Hordeum distichum

Linoleate hydroperoxide isomerase was mainly located in the embryos of barley grains and its activity decreased during germination. The enzyme partially purified from embryos converted 9-hydroperoxy, trans-10, cis-12-octadecadienoic acid to 9-hydroxy, 10-oxo, cis-12-octadecenoic acid and 13-hydroxy, 10-oxo, trans-11-octadecenoic acid in the ratio of ca 2:1.     

2,4-Trans-4,5-trans-4,5-dihydroxypipecolic acid and cis-5-hydroxypipecolic acid from leaves of Calliandra angustifolia and sap of C. Confusa

2,4-Trans-4,5-trans-4,5-dihydroxypipecolic acid and cis-5-hydroxypipecolic acid have been isolated from the leaves of Calliandra angustifolia and the sap of C. confusa. Distribution of these and other non-protein amino acids is discussed.     

Seasonal variations in trans-2-hexenal and linolenic acid in homogenates of Thea sinensis leaves

The seasonal variations in the amounts of C₆-volatile components cis-3-hexenal trans-2-hexenal n-hexanal) and their precursors (linoleic and linolenic acid) in homogenates of Thea sinensis leaves were quantitatively analyzed throughout the year. Formation of trans-2-hexenal began in the middle of April and reached a maximum during July. Towards autumn the aldehyde gradually decreased and, in winter (December to March), was virtually absent. The levels of cis-3-hexenol remained constant during May–December. cis-3-Hexenal showed a similar variation pattern to that of trans-2-hexenal. The major fatty acids in the leaves were palmitic, palmitoleic, oleic, linoleic and linolenic acid, and occurred in non-ionic lipids and phospholipid fractions. The amounts of linoleic and linolenic acid did not show any marked variation except for a big peak in October.     

Effect of cis-9,trans-11 and trans-10,cis-12 isomers of conjugated linoleic acid on the integrity and functionality of cryopreserved bovine spermatozoa

Plasma membranes of sperm subjected to low temperatures undergo changes in their structure and permeability. The addition of fatty acids in semen cryopreservation media may influence the sperm motility after thawing, possibly by maintaining the membrane fluidity due to their incorporation in lipid bilayers. In this work, different concentrations of the isomers cis-9,trans-11 and trans-10,cis-12 of conjugated linoleic acid (CLA) were added in the cryopreservation medium of bovine sperm. Four Jersey bulls were used, and the ejaculates were processed as a pool. The Tris-based extender (Dilutris®) was supplemented with 20% egg yolk (MB). The treatments with CLA (Luta-CLA®), which had oily presentation, were prepared from MB with addition of 1% sodium lauryl sulfate, and denominated MBL. The concentrations of CLA tested were 50, 100, and 150 μM. The motility characteristics of the post-thaw semen were analyzed by computerized analysis system (CASA), and plasma membrane integrity and acrosomal and mitochondrial function assessed by the association of the fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), JC-1 and Hoechst 33342. No significant differences were observed among treatments, excepting for a decreased mitochondrial potential of cells treated with 150 μM CLA. The addition of CLA, at the concentrations used, showed no advantages on the integrity and functionality of bovine sperm submitted to cryopreservation.     

Cis-monolignols in Fagus grandifolia and their possible involvement in lignification

Lignification in all plant species is assumed to occur exclusively via the dehydrogenative polymerization of the trans (E) monolignols, p-coumaryl, coniferyl and sinapyl alcohols. This assumption may have to be revised somewhat due to the presence of both E(trans) and Z (cis) isomers of p-hydroxy substituted cinnamic acids in grasses, and cis-coniferyl and cis-sinapyl alcohols in beech bark (Fagus grandifolia). This suggests that lignification of these tissues may use either cis- or trans-monolignols. By means of H₂O₂/peroxidase induction, we have prepared synthetic dehydrogenative polymerized (DHP) lignin from both cis- and trans-coniferyl alcohols. Under the tests employed, the degradation products from both DHPs were identical suggesting that either cis- or trans-monolignols are suitable substrates for this enzyme and, therefore, lignification. An alternative hypothesis is that the cis-monolignols accumulate in beech bark because they are not suitable substrates. Therefore, it would follow that the enzymes involved in lignification in vivo are highly specific.     

Intricacies of interactions,dynamics and solvent effects in enzyme catalysis: a computational perspective on cyclophilin A

The essential role of enzymes in biological processes has continually ignited sparks of interest in their mechanism of action. Fully understanding the mechanism of enzymes has broad implications in protein engineering and drug design. The more than five order of magnitude speed-up in the rate of peptidyl–prolyl cis–trans isomerisation by cyclophilin A (CypA) has been the target of intense research. CypA serves as a tractable model system, because it reversibly catalyses the rotation around peptidyl–prolyl bonds without any bond breakage or formation. Here, we discuss the results of recent computational approaches used to study the mechanism of CypA. We highlight the critical role of enzyme and substrate conformational dynamics in the developing interactions as the substrate approaches the transition state that results in an astonishing enhancement of isomerisation rate. The rate of isomerisation is affected by the intricate coupling between the dynamics of the substrate, enzyme and solvent. CypA binds its substrates via conformational selection, where rearrangements of key active site residues are necessary for substrate recognition. The conformational plasticity of the active site allows the enzyme to accommodate the most favourable interactions with the transition state that can be exploited for structure-based drug design.     

Enhancement of antibody synthesis in rats by feeding cis-9,trans-11 conjugated linoleic acid during early life

Previous studies have demonstrated that the intake of a 1% conjugated linoleic acid (CLA) diet in an 80:20 mixture of cis-9,trans-11 and trans-10,cis-12 exerts age-specific effects on the immune system: immunoglobulin enhancement and proliferative down-modulation in neonatal and adult rats, respectively. The present study evaluates the influence of the same diet on antibody synthesis of early infant Wistar rats during suckling and/or after weaning. Dietary supplementation was performed during suckling and early infancy (4 weeks), only during suckling (3 weeks), or only in early infancy (1 week). CLA content in plasma and serum immunoglobulin (Ig) G, IgM and IgA concentration were determined. Proliferation, cytokines and Ig production were evaluated on isolated splenocytes. Cis-9,trans-11- and trans-10,cis-12-CLA isomers were detected in the plasma of all CLA-supplemented animals, and the highest content was quantified in those rats supplemented over the longest period. These rats also exhibited higher concentrations of serum IgG, IgM and IgA. Moreover, splenocytes from CLA-supplemented rats showed the highest IgM and IgG synthesis and interleukin (IL)-6 production, whereas their proliferative ability was lower. In summary, in infant rats, we observed both the enhance antibody synthesis previously reported in neonates, and the reduced lymphoproliferation previously reported in adults.     

Thermal degradation and isomerisation kinetics of triolein studied by infrared spectrometry and GC-MS combined with chemometrics

Triolein, a triglyceride containing oleic acid as the only acid moiety in the glyceride molecules has been isothermally treated at 280, 300, and 325 °C in glass vials under nitrogen atmosphere. The products formed during the thermal treatment at each temperature have been analysed both by infrared spectrometry and GC-MS. The GC-MS analysis was performed after derivatisation of the fatty acids into their methyl esters (FAMEs).Chemometric tools were used in determining the concentrations of the main products namely triolein and trieaidin in the thermally treated mixtures. The concentration profiles of the trielaidin formed during thermal treatment at the above three temperatures were used in determining activation energy for the cis-trans isomerisation of triolein.The combined analysis reveals that the thermal treatment induces not only cis-trans isomerisation but also fission and fusion in the molecules. Furthermore, migration of the double bond in oleic and elaidic acids forming cis and trans isomers of the 18:1 acid was also observed. The heat-induced isomerisation in triolein follows a zeroth order reaction with an activation energy 41 ± 5 kcal/mol.     

Synthesis of 2-methylthio-cis- and trans-ribosylzeatin and their isolation from Pisum tRNA

The cis isomer of 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthiopurine and its 9-β- and 9-α-d-ribofuranosyl derivatives have been synthesized and their physical and spectroscopic properties are described. The biological activities of these compounds have been determined in the tobacco bioassay and are compared with those of 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-2-methylthiopurine and its β-ribofuranoside. The 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d-ribofuranosylpurine (ms-ribosylzeatin) isolated from a Pisum tRNA preparation was shown to consist of both isomers, which were separated by TLC and identified by comparisons of UV and MS with those of the synthetic compounds.