Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function

Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases.

Effect of altered dietary n-3 fatty acid intake upon plasma lipid fatty acid composition, conversion of [C-13]alpha-linolenic acid to longer-chain fatty acids and partitioning towards beta-oxidation in older men

The effect of increased dietary intakes of alpha-linolenic acid (ALNA) or eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for 2 months upon plasma lipid composition and capacity for conversion of ALNA to longer-chain metabolites was investigated in healthy men (52 (SD 12) years). After a 4-week baseline period when the subjects substituted a control spread, a test meal containing [U-C-13]ALNA (700 mg) was consumed to measure conversion to EPA, docosapentaenoic acid (DPA) and DHA over 48 h. Subjects were then randomised to one of three groups for 8 weeks before repeating the tracer study: (1) continued on same intake (control, n 5); (2) increased ALNA intake (10 g/d, n 4); (3) increased EPA+DHA intake (1.5 g/d, n 5). At baseline, apparent fractional conversion of labelled ALNA was: EPA 2.80, DPA 1.20 and DRA 0.04%. After 8 weeks on the control diet, plasma lipid composition and [C-13]ALNA conversion remained unchanged compared with baseline. The high-ALNA diet resulted in raised plasma triacylglycerol-EPA and -DPA concentrations and phosphatidylcholine-EPA concentration, whilst [C-13]ALNA conversion was similar to baseline. The high-(EPA+DHA) diet raised plasma phosphatidylcholine-EPA and -DHA concentrations, decreased [C-13]ALNA conversion to EPA (2-fold) and DPA (4-fold), whilst [C-13]ALNA conversion to DHA was unchanged. The dietary interventions did not alter partitioning of ALNA towards beta-oxidation. The present results indicate ALNA conversion was down-regulated by increased product (EPA+DHA) availability, but was not up-regulated by increased substrate (ALNA) consumption. This suggests regulation of ALNA conversion may limit the influence of variations in dietary n-3 fatty acid intake on plasma lipid compositions.

Electrochemical and photochemical conversion of [Ru3Ir(mu(3)-H)(CO)(13)] into [Ru3Ir(mu-H)(3)(CO)(12)]

Electrochemical and photochemical properties of the tetrahedral cluster [Ru3Ir(mu(3)-H)(CO)(13)] were studied in order to prove whether the previously established thermal conversion of this cluster into the hydrogenated derivative [Ru3Ir(mu-H)(3)(CO)(12)] also occurs by means of redox or photochemical activation. Two-electron reduction of [Ru3Ir(mu(3)-H)(CO)(13)] results in the loss of CO and concomitant formation of the dianion [Ru3Ir(mu(3)-H)(CO)(12)](2-). The latter reduction product is stable in CH2Cl2 at low temperatures but becomes partly protonated above 283 K into the anion [Ru3Ir(mu-H)(2)(CO)(12)](-) by traces of water. The dianion [Ru3Ir(mu(3)-H)(CO)(12)](2-) is also the product of the electrochemical reduction of [Ru3Ir(mu-H)(3)(CO)(12)] accompanied by the loss of H-2. Stepwise deprotonation of [Ru3Ir(mu-H)(3)(CO)(12)] with Et4NOH yields [Ru3Ir(mu-H)(2)(CO)(12)](-) and [Ru3Ir(mu(3)-H)(CO)(12)](2-). Reverse protonation of the anionic clusters can be achieved, e. g., with trifluoromethylsulfonic acid. Thus, the electrochemical conversion of [Ru3Ir(mu(3)-H)(CO)(13)] into [Ru3Ir(mu-H)(3)(CO)(12)] is feasible, demanding separate two-electron reduction and protonation steps. Irradiation into the visible absorption band of [Ru3Ir(mu3-H)(CO)(13)] in hexane does not induce any significant photochemical conversion. Irradiation of this cluster in the presence of CO with lambda(irr) > 340 nm, however, triggers its efficient photofragmentation into reactive unsaturated ruthenium and iridium carbonyl fragments. These fragments are either stabilised by dissolved CO or undergo reclusterification to give homonuclear clusters. Most importantly, in H-2-saturated hexane, [Ru3Ir(mu(3)-H)(CO)(13)] converts selectively into the [Ru3Ir(mu-H)(3)(CO)(12)] photoproduct. This conversion is particularly efficient at lambda(irr) > 340 nm.

Metal complexes with atropisomeric sulfur ligands in asymmetric hydroformylation X-ray structure of [Rh2(μ-biphes)(cod)2] (H2biphes = 4,4′-biphenanthrene-3,3′-dithiol)

The addition of the atropisomeric racemic sulfur compound 4,4′-biphenanthrene-3,3′-dithiol (H2 biphes) to a dichloromethane solution of [{M(μ-OMe)(cod)}2] (M = Rh, Ir, cod = cycloocta-1,5-diene) afforded the dithiolate-bridged complexes [{Rh2(μ-biphes)(cod)2}n] (n = 2 5 or n = 1 6) and [{Ir2(μ-biphes)(cod)2}n]·nCH2Cl27. When 1,1′-binaphthalene-2,2′-dithiol (H2 binas) reacted with [{Ir(μ-OMe)(cod)}2], complex [Ir2(μ-binas)(cod)2] 8 was obtained. Complexes 5 and 6 reacted with carbon monoxide to give the dinuclear tetracarbonyl complex [Rh2(μ-biphes)(CO)4] 9. The reaction of 9 with PR3 provided the mixed-ligand complexes [{Rh2(μ-biphes)(CO)2(PR3)2}2] · xCH2Cl2 (R = Ph, x = 2 10, C6H11, x = 1 11) and [{Rh2(μ-biphes)(CO)3(PR3)}2] · CH2Cl212 (R = OC6H4But-o). The crystal structure of 6 was determined by X-ray diffraction. Reaction of the dithioether ligand Me2biphes with [Rh(cod)2]ClO4 in CH2Cl2 solution afforded the cationic complex [Rh(cod)(Me2biphes)]ClO4 · CH2Cl213. Asymmetric hydroformylation of styrene was performed using the complexes described. The extent of aldehyde conversion ranges from 53 to 100%, with selectivities towards branched aldehydes in the range 51 to 96%. The enantioselectivities were quite low and did not exceed 20%.

Cis–trans isomerism in diphenoxido bridged dicopper complexes: role of crystallized water to stabilize the cis isomer, variation in magnetic properties and conversion of both into a trinuclear species

The trans-[Cu2L2Cl2] (1), and cis-[Cu2L2Cl2]·H2O (2) isomers of a diphenoxido bridged Cu2O2 core have been synthesized using a tridentate reduced Schiff base ligand 2-[(2-dimethylamino-ethylamino)-methyl]-phenol. The geometry around Cu(II) is intermediate between square pyramid and trigonal bipyramid (Addison parameter, tau = 0.463) in 1 but nearly square pyramidal (tau = 0.049) in 2. The chloride ions are coordinated to Cu(II) and are trans oriented in 1 but cis oriented in 2. Both isomers have been optimized using density functional theory (DFT) calculations and it is found that the trans isomer is 7.2 kcal mol(-1) more favorable than the cis isomer. However, the hydrogen bonding interaction of crystallized water molecule with chloride ions compensates for the energy difference and stabilizes the cis isomer. Both complexes have been converted to a very rare phenoxido-azido bridged trinuclear species, [Cu3L2(mu(1,1)-N-3)(2)(H2O)(2)(ClO4)(2)] (3) which has also been characterized structurally. All the complexes are antiferromagnetically coupled but the magnitude of the coupling constants are significantly different (J = -156.60, -652.31, and -31.54 cm(-1) for 1, 2, and 3 respectively). Density functional theory (DFT) calculations have also been performed to gain further insight into the qualitative theoretical interpretation on the overall magnetic behavior of the complexes.

A rare case of solution and solid state inter-conversion of two copper(II) dimers and a copper(II) chain

Three Cu(II)-azido complexes of formula [Cu2L2(N-3)(2)] (1), [Cu2L2(N-3)(2)]center dot H2O (2) and [CuL(N-3)](n) (3) have been synthesized using the same tridentate Schiff base ligand HL (2-[(3-methylaminopropylimino)-methyl]-phenol), the condensation product of N-methyl-1,3-propanediamine and salicyldehyde). Compounds 1 and 2 are basal-apical mu-1,1 double azido bridged dimers. The dimeric structure of 1 is centro-symmetric but that of 2 is non-centrommetric. Compound 3 is a mu-1,1 single azido bridged 1D chain. The three complexes interconvert in solution and can be obtained in pure form by carefully controlling the synthetic conditions. Compound 2 undergoes an irreversible transformation to 1 upon dehydration in the solid state. The magnetic properties of compounds 1 and 2 show the presence of weak antiferromagnetic exchange interactions mediated by the double 1,1-N-3 azido bridges (J = -2.59(4) and -0.10(1) cm-(1), respectively). The single 1,1-N-3 bridge in compound 3 mediates a negligible exchange interaction.

Rethinking the Conversion of Scotland in the fifth and sixth centuries

Reconsidering the initial Christian Conversion of Scotland in the fifth and sixth centuries AD, using archaeological and historical evidence, it is argued that this was carried out by missionaries from what had been Roman Britain. It is shown that this missionary activity - and similar British missions in Ireland - represents the first instance of Western missionary work beyond the former Roman imperial frontiers. The location of the northern frontier of Roman Britain in the fourth century, and the meaning of Pictish Class 1 symbol stones, are discussed as part of the broader argument.

Reversible, nondegradative conversion of crystalline aromatic poly(ether ketone)s into organo-soluble poly(ether dithioketal)s

Crystalline aromatic poly(ether ketone)s Such as PEEK and PEK may be cleanly and reversibly derivatized by dithioketalization of the carbonyl groups With 1,2-ethanedithiol or 1,3-propanedithiol under strong acid conditions. The resulting 1,3-dithiolane and 1,3-dithiane polymers are hydrolytically stable, amorphous, and readily soluble in organic solvents such as chloroform and THF and are thus (unlike their parent polymers) easily characterized by gel permeation chromatography (GPC). GPC analysis of a range of derivatized PEEK samples using light-scattering detection revealed, in some instances, a bimodal molecular weight distribution with a small but potentially significant (and previously undetected) very high-molecular-weight fraction.

Some new strategies for the chemical separation of actinides and lanthanides

A future goal in nuclear fuel reprocessing is the conversion or transmutation of the long-lived radioisotopes of minor actinides, such as americium, into short-lived isotopes by irradiation with neutrons. In order to achieve this transmutation, it is necessary to separate the minor actinides(III), [An(Ill)], from the lanthanides(III), [Ln(Ill)], by solvent extraction (partitioning), because the lanthanides absorb neutrons too effectively and hence limit neutron capture by the transmutable actinides. Partitioning using ligands containing only carbon, hydrogen, nitrogen and oxygen atoms is desirable because they are completely incinerable and thus the final volume of waste is minimised [1]. Nitric acid media will be used in the extraction experiments because it is envisaged that the An(III)/Ln(III) separation process could take place after the PUREX process. There is no doubt that the correct design of a molecule that is capable of acting as a ligand or extraction reagent is required for the effective separation of metal ions such as actinides(III) from lanthanides. Recent attention has been directed towards heterocyclic ligands with for the preferential separation of the minor actinides. Although such molecules have a rich chemistry, this is only now becoming sufficiently well understood in relation to the partitioning process [2]. The molecules shown in Figures I and 2 will be the principal focus of this study. Although the examples chosen here are used rather specific, the guidelines can be extended to other areas such as the separation of precious metals [3].

Influence of plant and bacterial myrosinase activity on the metabolic fate of glucosinolates in gnotobiotic rats

The breakdown of glucosinolates, a group of thioglucoside compounds found in cruciferous plants, is catalysed by dietary or microbial myrosinase. This hydrolysis releases a range of breakdown products among which are the isothiocyanates, which have been implicated in the cancer-protective effects of cruciferous vegetables. The respective involvement of plant myrosinase and gut bacterial myrosinase in the conversion, in vivo, of glucosinolates into isothiocyanates was investigated in sixteen Fischer 344 rats. Glucosinolate hydrolysis in gnotobiotic rats harbouring a whole human faecal flora (Flora+) was compared with that in germ-free rats (Flora-). Rats were offered a diet where plant myrosinase was either active (Myro+) or inactive (Myro-). The conversion of prop-2-enyl glucosinolate and benzyl glucosinolate to their related isothiocyanates, allyl isothiocyanate and benzyl isothiocyanate, was estimated using urinary mercapturic acids, which are endproducts of isothiocyanate metabolism. The highest excretion of urinary mercapturic acids was found when only plant myrosinase was active (Flora-, Myro+ treatment). Lower excretion was observed when both plant and microbial myrosinases were active (Flora+, Myro+ treatment). Excretion of urinary mercapturic acids when only microbial myrosinase was active (Flora+, Myro- treatment) was low and comparable with the levels in the absence of myrosinase (Flora-, Myro- treatment). No intact glucosinolates were detected in the faeces of rats from the Flora+ treatments confirming the strong capacity of the microflora to break down glucosinolates. The results confirm that plant myrosinase can catalyse substantial release of isothiocyanates in vivo. The results also suggest that the human microflora may, in some circumstances, reduce the proportion of isothiocyanates available for intestinal absorption.

Potential prebiotic activity of oligosaccharides obtained by enzymatic conversion of durum wheat insoluble dietary fibre into soluble dietary fibre

Background and aims: Epidemiological evidence indicates that cereal dietary fibre (DF) may have several cardiovascular health benefits. The underlying mechanisms have not yet been elucidated. Here, the potential nutritional effects of physico-chemical. properties modifications of durum wheat dietary fibre (DWF) induced by enzyme treatment have been investigated. Methods and results: The conversion of the highly polymerised insoluble dietary fibre into soluble feruloyl oligosaccharides of DWF was achieved by a tailored enzymatic treatment. The in vitro fermentation and release of ferulic acid by intestinal microbiota from DWF before and after the enzymatic treatment were assessed using a gut model validated to mimic the human colonic microbial environment. Results demonstrated that, compared to DWF, the enzyme-treated DWF (ETD-WF) stimulated the growth of bifidobacteria and lactobacilli. Concurrently, the release of free ferulic acid by ET-DWF was almost three times higher respect to the control. No effect on the formation of short chain fatty acids was observed. Conclusions: The conversion of insoluble dietary fibre from cereals into soluble dietary fibre generated a gut microbial fermentation that supported bifidobacteria and lactobacilli. The concurrent increase in free ferulic acid from the enzyme-treated DWF might result in a higher plasma ferulic acid concentration which could be one of the reasons for the health benefits reported for dietary fibre in cardiovascular diseases. (c) 2008 Elsevier B.V. All rights reserved.

The influence of dietary fatty acid composition on N-ethyl-Nnitrosourea-induced mammary tumour incidence in the rat and on the composition of inositol- and ethanolamine-phospholipids of normal and tumour mammary tissue

This study has investigated the influence of dietary fatty acid composition on mammary tumour incidence in N-ethyl-N-nitrosourea (ENU)-treated rats and has compared the susceptibility to dietary fatty acid modification of the membrane phospholipids phosphatidyliuositol (PI) and phosphatidylethanolamine (PE) from normal and tumour tissue of rat mammary gland. The incidence of mammary tumours was significantly lower in fish oil- (29%), compared with olive oil- (75%; P < 0.04) but not maize oil- (63%; P < 0.1) fed animals. No differences in PI fatty acid composition were found in normal or tumour tissue between rats fed on maize oil, olive oil or fish oil in diets from weaning. When normal and tumour tissue PI fatty acids were compared, significantly higher amounts of stearic acid (18:O) were found in tumour than normal tissue in rats given olive oil (P < 0.05). A similar trend was found in animals fed on maize oil, although differences between normal and tumour tissue did not reach a level of statistical significance (P < 0.1). In mammary PE, maize oil-fed control animals had significantly higher levels of linoleic acid (18:2n-6) than either olive oil- or fish oil-fed animals (P < 0.05, both cases) and levels of arachidonic acid were also higher in maize oil- compared with fish oil-fed animals (P < 0.05). In tumourbearing animals no differences in PE fatty acid composition were found between the three dietary groups. When normal and tumour tissue PE fatty acids were compared, significantly lower amounts of liuoleic acid (18:2n-6; P < 0.01) and significantly greater amounts of arachidonic acid (20:4n-6; P < 0.05) were found in tumour than normal tissue of rats fed on maize oil. The present study shows that the fatty acid composition of PI from both normal and tumour tissue of the mammary gland is resistant to dietary fatty acid modification. The PE fraction is more susceptible to dietary modification and in this fraction there is evidence of increased conversion of linoleic acid to arachidonic acid in tumour compared with normal tissue. Lower tumour incidence rates in rats given fish oils may in part be due to alteration in prostanoid metabolism secondary to displacement of arachidonic acid by eicosapentaenoic acid, but PE rather than PI would appear to be the most likely locus for diet-induced alteration in prostanoid synthesis in this tissue. Effects of dietary fatty acids other than on the balance of n-6 and n-3 fatty acids, and on prostanoid metabolism, should also be considered. The significance of increased stearic acid content of PI in tumours of olive oil-fed animals and the possible influence of dietary fatty acids on the capacity for stearic acid accumulation requires further study.

Equol: a comparison of the effects of the racemic compound with that of the purified S-enantiomer on the growth, invasion, and DNA integrity of breast and prostate cells in vitro

It has been postulated that the R- and S-equol enantiomers have different biological properties given their different binding affinities for the estrogen receptor. S-(-)equol is produced via the bacterial conversion of the soy isoflavone daidzein in the gut. We have compared the biological effects of purified S-equol to that of racemic (R and S) equol on breast and prostate cancer cells of varying receptor status in vitro. Both racemic and S-equol inhibited the growth of the breast cancer cell line MDA-MB-231 (> or = 10 microM) and the prostate cancer cell lines LNCaP (> or = 5 microM) and LAPC-4 (> or = 2.5 microM). The compounds also showed equipotent effects in inhibiting the invasion of MDA-MB-231 and PC-3 cancer cells through matrigel. S-equol (1, 10, 30 microM) was unable to prevent DNA damage in MCF-7 or MCF-10A breast cells following exposure to 2-hydroxy-4-nonenal, menadione, or benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide. In contrast, racemic equol (10, 30 microM) prevented DNA damage in MCF-10A cells following exposure to 2-hydroxy-4-nonenal or menadione. These findings suggest that racemic equol has strong antigenotoxic activity in contrast to the purified S-equol enantiomer implicating the R-, rather than the S-enantiomer as being responsible for the antioxidant effects of equol, a finding that may have implications for the in vivo chemoprotective properties of equol.