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Ionic liquids, also called molten salt, are mixtures of cations and anions that melt below 100℃. Typical ionic liquids are dialkylimidazolium cations with weakly coordinating anions such as (MeOSO₃) or (PF∧6). Advanced ionic liquids such as choline citrate have biodegradable, less expensive, and less toxic anions and cations. Deep eutectic solvents are also included in the advanced ionic liquids. Deep eutectic solvents are mixtures of salts such as choline chloride and uncharged hydrogen bond donors such as urea, oxalic acid, or glycerol. For example, a mixture of choline chloride and urea in 1:2 molar ratio liquefies to form a deep eutectic solvent. Their properties are similar to those of ionic liquids. Water-miscible ionic liquids as cosolvents with water enhance the solubility of substrates or products. Although traditional water-miscible organic solvents also enhance solubility, they often inactivate enzymes, while ionic liquids do not. The enhanced solubility of substrates can increase the rate of reaction and often increases the regio- or enantioselectivity. Ionic liquids can also be solvents for non-aqueous reactions. In these cases, they are especially suited to dissolve polar substrates. Polar organic solvent alternatives inactivate enzymes, but ionic liquids do not even when they have similar polarities. Besides their solubility properties, ionic liquids and deep eutectic solvents may be greener than organic solvents because ionic liquids are nonvolatile, and can be made from nontoxic components. This review covers selected examples of enzyme catalyzed reaction in ionic liquids that demonstrate their advantages and unique properties, and point out opportunities for new applications. Most examples involve hydrolases, but oxidoreductases and even whole cell reactions have been reported in ionic liquids.

A three-dimensional structure for the monomeric iron-containing hydrogenase (Cpl) from Clostridium pasteurianum was determined to 1.8 angstrom resolution by X-ray crystallography using multiwavelength anomalous dispersion (MAD) phasing. Cpl, an enzyme that catalyzes the two-electron reduction of two protons to yield dihydrogen, was found to contain 20 gram atoms of iron per mole of protein, arranged into five distinct [Fe-S] clusters. The probable active-site cluster, previously termed the H-cluster, was found to be an unexpected arrangement of six iron atoms existing as a [4Fe-4S] cubane subcluster covalently bridged by a cysteinate thiol to a [2Fe] subcluster. The iron atoms of the [2Fe] subcluster both exist with an octahedral coordination geometry and are bridged to each other by three non-protein atoms, assigned as two sulfide atoms and one carbonyl or cyanide molecule. This structure provides insights into the mechanism of biological hydrogen activation and has broader implications for [Fe-S] cluster structure and function in biological systems

Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing.

Reactive oxygen species are believed to perform multiple roles during plant defense and possibly as cellular signaling molecules. In animals, nitric oxide (NO) is an important redox-active signaling molecule. Here we show that infection of resistant, but not susceptible, tobacco with tobacco mosaic virus resulted in enhanced NO synthase (NOS) activity. Furthermore, administration of NO donors or recombinant mammalian NOS to tobacco plants or tobacco suspension cells triggered expression of the defense-related genes encoding pathogenesis-related 1 protein and phenylalanine ammonia lyase (PAL). These genes were also induced by cyclic GMP (cGMP) and cyclic ADP-ribose, two molecules that can serve as second messengers for NO signaling in mammals. Consistent with cGMP levels. Furthermore, NO-induced activation of PAL was blocked by 6-anilino-5,8-quinolinedione and 1H-(1,2,4)-oxadizole[4,3-alpha]quinoxalin-1-one, two inhibitors of guanylate cyclase. Although 6-anilino-5,8-quinolinedione fully blocked PAL activation, inhibition by 1H-(1,2,4)-oxadiozole[4,3-alpha]quinoxalin-1-one was not entirely complete, suggesting the existence of cGMP-independent, as well as cGMP-dependent, NO signaling. We conclude that several critical players of animal NO signaling are also operative in plants

A highly active synthetic auxin response element (AuxRE), referred to as DR5, was created by performing site-directed mutations in a natural composite AuxRE found in the soybean GH3 promoter. DR5 consisted of tandem direct repeats of 11 bp that included the auxin-responsive TGTCTC element. The DR5 AuxRE showed greater auxin responsiveness than a natural composite AuxRE and the GH3 promoter when assayed by transient expression in carrot protoplasts or in stably transformed Arabidopsis seedlings, and it provides a useful reporter gene for studying auxin-responsive transcription in wild-type plants and mutants. An auxin response transcription factor, ARF1, bound with specificity to the DR5 AuxRE in vitro and interacted with Aux/IAA proteins in a yeast two-hybrid system. Cotransfection experiments with natural and synthetic AuxRE reporter genes and effector genes encoding Aux/IAA proteins showed that overexpression of Aux/IAA proteins in carrot protoplasts resulted in specific repression of TGTCTC AuxRE reporter gene expression

A conserved regulatory mechanism protects plants against the potentially damaging effects of excessive light. Nearly all photosynthetic eukaryotes are able to dissipate excess absorbed light energy in a process that involves xanthophyll pigments. To dissect the role of xanthophylls in photoprotective energy dissipation in vivo, we isolated Arabidopsis xanthophyll cycle mutants by screening for altered nonphotochemical quenching of chlorophyll fluorescence. The npq1 mutants are unable to convert violaxanthin to zeaxanthin in excessive light, whereas the npq2 mutants accumulate zeaxanthin constitutively. The npq2 mutants are new alleles of aba1, the zeaxanthin epoxidase gene. The high levels of zeaxanthin in npq2 affected the kinetics of induction and relaxation but not the extent of nonphotochemical quenching. Genetic mapping, DNA sequencing, and complementation of npq1 demonstrated that this mutation affects the structural gene encoding violaxanthin deepoxidase. The npq1 mutant exhibited greatly reduced nonphotochemical quenching, demonstrating that violaxanthin deepoxidation is required for the bulk of rapidly reversible nonphotochemical quenching in Arabidopsis. Altered regulation of photosynthetic energy conversion in npq1 was associated with increased sensitivity to photoinhibition. These results, in conjunction with the analysis of npq mutants of Chlamydomonas, suggest that the role of the xanthophyll cycle in nonphotochemical quenching has been conserved, although different photosynthetic eukaryotes rely on the xanthophyll cycle to different extents for the dissipation of excess absorbed light energy

▪ Abstract  Desaturation of a fatty acid first involves the enzymatic removal of a hydrogen from a methylene group in an acyl chain, a highly energy-demanding step that requires an activated oxygen intermediate. Two types of desaturases have been identified, one soluble and the other membrane-bound, that have different consensus motifs. Database searching for these motifs reveals that these enzymes belong to two distinct multifunctional classes, each of which includes desaturases, hydroxylases, and epoxidases that act on fatty acids or other substrates. The soluble class has a consensus motif consisting of carboxylates and histidines that coordinate an active site diiron cluster. The integral membrane class contains a different consensus motif composed of histidines. Biochemical and structural similarities between the integral membrane enzymes suggest that this class also uses a diiron cluster for catalysis. Soluble and membrane enzymes have been successfully re-engineered for substrate specificity and reaction outcome. It is anticipated that rational design of these enzymes will result in new and desired activities that may form the basis for improved oil crops.

Like obese humans, Zucker diabetic fatty (ZDF) rats exhibit early β cell compensation for insulin resistance (4-fold β cell hyperplasia) followed by decompensation (>50% loss of β cells). In prediabetic and diabetic ZDF islets, apoptosis measured by DNA laddering is increased 3- and >7-fold, respectively, compared with lean ZDF controls. Ceramide, a fatty acid-containing messenger in cytokine-induced apoptosis, was significantly increased (P < 0.01) in prediabetic and diabetic islets. Free fatty acids (FFAs) in plasma are high (>1 mM) in prediabetic and diabetic ZDF rats; therefore, we cultured prediabetic islets in 1 mM FFA. DNA laddering rose to 19.6% vs. 4.6% in lean control islets, preceded by an 82% increase in ceramide. C2-Ceramide without FFA induced DNA laddering, but fumonisin B1, a ceramide synthetase inhibitor, completely blocked FFA-induced DNA laddering in cultured ZDF islets. [3H]Palmitate incorporation in [3H]ceramide in ZDF islets was twice that of controls, but [3H]palmitate oxidation was 77% less. Triacsin C, an inhibitor of fatty acyl-CoA synthetase, and troglitazone, an enhancer of FFA oxidation in ZDF islets, both blocked DNA laddering. These agents also reduced inducible nitric oxide (NO) synthase mRNA and NO production, which are involved in FFA-induced apoptosis. In ZDF obesity, β cell apoptosis is induced by increased FFA via de novo ceramide formation and increased NO production.

Glucocorticoid hormones, acting via nuclear receptors, regulate many metabolic processes, including hepatic gluconeogenesis. It recently has been recognized that intracellular glucocorticoid concentrations are determined not only by plasma hormone levels, but also by intracellular 11 beta-hydroxysteroid dehydrogenases (11 beta-HSDs), which interconvert active corticosterone (cortisol in humans) and inert 11-dehydrocorticosterone (cortisone in humans). 11 beta-HSD type 2, a dehydrogenase, thus excludes glucocorticoids from otherwise nonselective mineralocorticoid receptors in the kidney. Recent data suggest the type 1 isozyme (11 beta-HSD-1) may function as an 11 beta-reductase, regenerating active glucocorticoids from circulating inert 11-keto forms in specific tissues, notably the liver. To examine the importance of this enzyme isoform in vivo, mice were produced with targeted disruption of the 11 beta-HSD-1 gene. These mice were unable to convert inert 11-dehydrocorticosterone to corticosterone in vivo. Despite compensatory adrenal hyperplasia and increased adrenal secretion of corticosterone, on starvation homozygous mutants had attenuated activation of the key hepatic gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, presumably, because of relative intrahepatic glucocorticoid deficiency. The 11 beta-HSD-1 -/- mice were found to resist hyperglycemia provoked by obesity or stress. Attenuation of hepatic 11 beta-HSD-1 may provide a novel approach to the regulation of gluconeogenesis

Induction of phase 2 detoxication enzymes [e.g., glutathione transferases, epoxide hydrolase, NAD(P)H: quinone reductase, and glucuronosyltransferases] is a powerful strategy for achieving protection against carcinogenesis, mutagenesis, and other forms of toxicity of electrophiles and reactive forms of oxygen. Since consumption of large quantities of fruit and vegetables is associated with a striking reduction in the risk of developing a variety of malignancies, it is of interest that a number of edible plants contain substantial quantities of compounds that regulate mammalian enzymes of xenobiotic metabolism. Thus, edible plants belonging to the family Cruciferae and genus Brassica (e.g., broccoli and cauliflower) contain substantial quantities of isothiocyanates (mostly in the form of their glucosinolate precursors) some of which (e.g., sulforaphane or 4-methylsulfinylbutyl isothiocyanate) are very potent inducers of phase 2 enzymes. Unexpectedly, 3-day-old sprouts of cultivars of certain crucifers including broccoli and cauliflower contain 10-100 times higher levels of glucoraphanin (the glucosinolate of sulforaphane) than do the corresponding mature plants. Glucosinolates and isothiocyanates can be efficiently extracted from plants, without hydrolysis of glucosinolates by myrosinase, by homogenization in a mixture of equal volumes of dimethyl sulfoxide, dimethylformamide, and acetonitrile at -50 degrees C. Extracts of 3-day-old broccoli sprouts (containing either glucoraphanin or sulforaphane as the principal enzyme inducer) were highly effective in reducing the incidence, multiplicity, and rate of development of mammary tumors in dimethylbenz(a)anthracene-treated rats. Notably, sprouts of many broccoli cultivars contain negligible quantities of indole glucosinolates, which predominate in the mature vegetable and may give rise to degradation products (e.g., indole-3-carbinol) that can enhance tumorigenesis. Hence, small quantities of crucifer sprouts may protect