Purification and properties of hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase from potatoes

A rapid method for the purification of hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (CQT) from potato tubers which had been stored at low temperatures is described. The method involves affinity chromatography on Blue Sepharose with biospecific desorption of CQT with its substrate, CoA. Elution of the Blue Sepharose column with a gradient of CoA leads to the resolution of CQT, a protein with MW of ca 41500, into 3 peaks of activity; the largest peak elutes first. This fraction is purified × 1440 and gives a single band of protein after PAGE which suggests a high degree of purity. The properties of the 3 fractions of CQT, with respect to substrates and to a number of inhibitors, are described. The first and last eluting CQT fractions are specific for quinate and show no activity towards shikimate. The second peak, however, shows a small activity towards shikimate but this is thought to be due to an underlying peak of a shikimate specific enzyme. The major peak of CQT activity found in potatoes stored at 0° is absent from those stored at 10° throughout the period after harvest.     

The enzymic conversion of hydroxycinnamic acids to p-coumarylquinic and chlorogenic acids in tomato fruits

Two enzymes thought to be involved in the biosynthesis of chlorogenic acid have been separated and purified by ion exchange chromatography and their properties studied. These two enzymes, p-coumarate CoA ligase and hydroxycinnamyl CoA: quinate hydroxycinnamyl transferase, acting together catalyse the conversion of p-coumaric acid to 5′-p-coumarylquinic acid and of caffeic acid to chlorogenic acid. The ligase has a higher affinity for p-coumaric than for caffeic acid and will in addition activate a number of other cinnamic acids such as ferulic, isoferulic and m-coumaric acids but not cinnamic acid. The transferase shows higher activity and affinity with p-coumaryl CoA than caffeyl CoA. It also acts with ferulyl CoA but only very slowly. The enzyme shows high specificity for quinic acid; shikimic acid is esterified at only 2% of the rate with quinic acid and glucose is not a substrate. The transferase activity is reversible and both chlorogenic acid and 5′-p-coumarylquinic acids are cleaved in the presence of CoA to form quinic acid and the corresponding hydroxycinnamyl CoA thioester.     

Changes in the activity of enzymes of phenylpropanoid metabolism in tomatoes stored at low temperatures

A large increase in the activity of an enzyme involved in chlorogenic acid metabolism, hydroxycinnamyltransferase occurs in tomatoes stored at low temperatures. In contrast, the activity of the enzyme remains constant or falls slightly during normal ripening at 20°. The rise in activity occurs at temperatures below 10° and fails to occur at 15° or 20°. This increase in activity during low temperature storage occurs with fruit at all stages of ripening from mature green to fully ripe. The hydroxycinnamyltransferase of chilled tomatoes falls rapidly on transfer to 20° with a lag of about 4–8 hr and within 48 hr returns to that of unchilled fruit. The effects of such warming treatments are reversible since when a chilling period is resumed following warming to 20°, the rise in hydroxycinnamyltransferase activity is also resumed. Of the 5 other enzymes of phenylpropanoid metabolism studied, only PAL shows a similar increase in activity during low temperature storage although the activity of the other enzymes was maintained at higher levels in fruit at 2° than at 20°. The possible relationship between the behaviour of hydroxycinnamyltransferase activity at various temperatures and the known susceptibility of tomatoes to chilling injury is discussed.     

Photocontrol of chlorogenic acid biosynthesis in potato tuber discs

The appearance of phenylalanine ammonia-lyase activity and the accumulation of chlorogenic acid in potato tuber discs are stimulated by illumination with white light, whereas the appearance of cinnamic acid 4-hydroxylase activity is unaffected by illumination. The photosensitive step in chlorogenic acid biosynthesis may be by-passed by treatment of discs with exogenous supplies of cinnamic acid, whereas treatment of discs with phenylalanine does not isolate the photosensitive step. Therefore, the site of photocontrol of chlorogenic acid biosynthesis in potato tuber discs is the reaction catalysed by phenylalanine ammonia-lyase. Cinnamic acid 4-hydroxylase activity in vitro is unaffected by p-coumaric acid, caffeic acid or chlorogenic acid. Phenylalanine ammonia-lyase activity in vitro is sensitive to inhibition by cinnamic acid. The in vitro properties of the two enzymes are also consistent with the hypothesis that phenylalanine ammonia-lyase rather than cinnamic acid 4-hydroxylase is important in the regulation of chlorogenic acid biosynthesis in potato tuber discs.     

Changes in the amounts of chlorogenic and caffeic acids during tobacco seed maturation

During maturation of tobacco seed, the amount of chlorogenic acid rapidly increased between days 7 and 11 after anthesis and then decreased until day 15. Chlorogenic acid was not detected in fully mature seed. On the other hand, caffeic acid appeared from day 15, reached a maximum at day 21, and rapidly disappeared by day 25 although the amount was always low in comparison with that of chlorogenic acid. Changes in dry wt, water content, testa color and germination of tobacco seeds during maturation were also investigated. The results suggested that there were rapid biochemical changes in seed between days 11 and 15 after anthesis.     

The fate of the phytoalexin ipomeamarone: furanoterpenes and butenolides from Ceratocystis fimbriata-infected sweet potatoes

Nine new sesquiterpenes related biosynthetically to ipomeamarone, the well-known sweet potato phytoalexin, have been isolated from Ceratocystis fimbriata-infected sweet potato root tissue and the structures determined. Their biosynthetic relationship with previously identified furanoterpenes is discussed.     

Kinetic study of possible intermediates in the biosynthesis of chlorogenic acid in Cestrum poeppigii

p-Coumaric and 3-O-p-coumarylquinic acid seem to be important precursors of chlorogenic acid in the leaves of Cestrum poeppigii. 3-O-Cinnamylquinic acid, which has a very small metabolic activity, is of little importance in this respect. The kinetics of incorporation of radioactivity from t-cinnamic acid-3-[¹⁴C] into p-coumaric, 3-O-p-coumarylquinic, chlorogenic and 3-O-cinnamylquinic acid showed that the biosynthetic rates for these products decrease in the order shown. For p-coumaric acid, which has a markedly high metabolic activity, a turnover rate of 28 μg/hr and per gram fresh plant leaf, was calculated. Some trapping experiments with caffeic acid, and the acids mentioned above and using either t-cinnamic acid-3-[¹⁴C] or p-coumaric acid-2-[¹⁴C] as precursor, are discussed. A HPLC method for the rapid determination of phenolic acids in plant extracts, is described.     

Interaction of salicylate and ibuprofen with the carboxylic acid: CoA ligases from bovine liver mitochondria

Neither salicylate nor ibuprofen was a substrate or inhibitor of the long-chain fatty acid: CoA ligase. In contrast, all three xenobiotic-metabolizing medium-chain fatty acid:CoA ligases (XL-I, XL-II, and XL-III) had activity toward salicylate. The K_m value for salicylate was similar for all three forms (2 to 3 μM), but XL-II and XL-III had higher activity at V_max. For ibuprofen, only XL-III catalyzed its activation, and it had a K_m for ibuprofen of 36 μM. Studies of salicylate inhibition of XL-I, XL-II, and XL-III revealed that it inhibited the benzoate activity of all three forms with K₁ values of ca. 2 μM, which is in agreement with the K_m values obtained with salicylate as substrate. Kinetic analysis revealed that salicylate conjugation by all three forms is characterized by substrate inhibition when salicylate exceeds ca. 20 μM. Substrate inhibition was more extensive with XL-I and XL-III. Previous work on the ligases employed assay concentrations of salicylate in the range of 0.1 to 1.0 mM, which are clearly inhibitory, particularly toward XL-I and XL-III. Thus, activity was not properly measured in previous studies, which accounts for the fact that salicylate conjugation was only found with one form, which is most likely XL-II since it has the highest V_max activity and shows the least amount of substrate inhibition. Studies with ibuprofen indicated that it inhibited XL-I, XL-II, and XL-III, with K₁ values being in the range of 75–125 μM. The short-chain ligase was inhibited by both salicylate and ibuprofen with K₁ values of 93 and 84 μM, respectively. It was concluded that pharmacological doses of salicylate, but not ibuprofen, will affect the metabolism of medium-chain fatty acids and carboxylic acid xenobiotics and that the previously described mitochondrial ibuprofen:CoA ligase activity is attributable to XL-III. © 1996 John Wiley & Sons, Inc.     

Single-laboratory validation for the determination of caffeic acid and seven caffeoylquinic acids in sweet potato leaves

A single-laboratory validation study was conducted on an HPLC method for the detection and quantification of caffeic acid (CA) and seven species of caffeoylquinic acids (CQAs) in lyophilized sweet potato leaves. The procedure for extraction of the analytes from the matrix and the HPLC conditions for the efficient separation of CA and CQAs were optimized. In the proposed method, a relative response factor to one of the CQAs (5-CQA) was used to quantify the others. The method performed well in terms of precision when carried out on five different days and demonstrated Horwitz ratio (HorRat) scores ranging from 0.5 to 1.0 for all analytes, which were well within the limits of performance acceptability. Accuracy testing at three levels showed an overall recovery of 94% when duplicated on five different days. Moreover, a stability study revealed that all analytes in both standard solution and sample extract were stable for 28?days.     

Changes in bulk protein of tobacco leaves on aerobic autolysis: Hydrogenation studies and identification of bound quinic acid

During aerobic autolysis and in commercial curing, the bulk proteins of tobacco leaves become coupled with quinic acid, presumably in consequence of coupling of chlorogenic acid congeners with lysine ε-NH₂ groups. Quinic acid derivatives, prepared from acid hydrolysates of such altered proteins, were identified by GC-MS. Such proteins were also hydrogenated over Rh/Al₂O₃ with a view to stabilizing the hypothetical linkages. Difficulties in removing contaminant Al had to be overcome. Evidence was then obtained (by GLC of derivatives) for several components, in acid hydrolysates of hydrogenated altered proteins, which were neither normal hydrogenation products of the common amino acids nor derivatives of quinic acid. Details of the chromatograms and mass spectra of quinic acid derivatives are provided in a supplementary publications.     

Effect of repeated harvesting on the content of caffeic acid and seven species of caffeoylquinic acids in sweet potato leaves

The purpose of this study was to investigate the effect of repeated harvesting on the content of caffeic acid (CA) and seven species of caffeoylquinic acids (CQAs) in sweet potato leaves using a newly developed high-performance liquid chromatography method. Six cultivars and two breeding lines were used in this study. Leaves were collected at monthly intervals from 1st harvest (May) to 4th harvest (August) in 2011 and 2012. ANOVA analysis revealed that the contents of CQAs were significantly different among all cultivars and breeding lines, but no significant differences were found for CA. No annual variation was confirmed in CA and CQAs. Repeated harvest of sweet potato leaves affected the content of only 4-CQA and 5-CQA. Post-hoc comparisons using Tukey’s method indicated that the contents of 4-CQA and 5-CQA in sweet potato leaves harvested at first time were significantly higher compared to those at the other harvest times.     

Antidiabetic and antihyperlipidemic activity of asiatic acid in diabetic rats,role of HMG CoA: In vivo and in silico approaches

Hyperlipidemia is an associated complication of diabetes and also a major risk factor for cardiovascular diseases. The present study was designed to examine the antihyperlipidemic effect of asiatic acid (AA) in streptozotocin (STZ) induced diabetic rats. Diabetes was induced in male Wistar rats by a single intraperitoneal injection of STZ (40 mg/kg b.w.). Diabetic rats show increased plasma glucose, total cholesterol, triglycerides, free fatty acids, phospholipids, low density lipoprotein, very low density liprotein, atherogenic index and decreased insulin and high density lipoprotein in diabetic rats. The activity of 3-hydroxy 3-methylglutaryl coenzyme A (HMG CoA) reductase increased significantly in contrast to the activities of lipoprotein lipase and lecithin cholesterol acyltransferase. In addition, the molecular docking of AA against HMG CoA reductase involved in cholesterol biosynthesis using Argus software. Diabetic rats were treated with AA shifted all these parameters towards normalcy. AA has shown best ligand binding energy 11.8122 kcal/mol. The antihyperlipidemic effect of AA was compared with glibenclamide; a well-known antihyperglycemic drug. In conclusion, this study indicates that AA showed an antihyperlipidemic effect in addition to its antidiabetic effect in experimental diabetes.     

Changes in phenylalanine ammonia-lyase levels in excised potato tuber tissue: effects of the gaseous environment and desensitization to cinnamate repression by in situ pre-incubation

Abstract. The rise in phenylalanine ammonia-lyase (PAL) activity following excision of potato tuber discs is antagonized by increasing partial pressures of CO₂ This inhibition is potentiated by depleting the atmospheric ethylene level. We suggest that the previously observed suppression of PAL appearance by in situ incubation of excised discs in reassembled tubers may be related to an internal atmosphere relatively rich in CO₂ and of low ethylene content. The transition to an oscillatory time course of PAL activity that follows transfer of discs from in situ incubation to air appears to be accompanied by the development of enzyme activity becoming desensitized to repression by exogenous cinnamate. The concentration dependence of cinnamate uptake is not significantly altered by in situ pre-incubation of tuber discs.     

Farnesoic acid O-methyl transferase (FAMeT) isoforms: conserved traits and gene expression patterns related to caste differentiation in the stingless bee, Melipona scutellaris

Farnesoic acid O-methyl transferase (FAMeT) is the enzyme that catalyzes the formation of methyl farnesoate (MF) from farnesoic acid (FA) in the biosynthetic pathway of juvenile hormone (JH). This work reports the cloning, sequencing, and expression of FAMeT gene from the stingless bee Melipona scutellaris (MsFAMeT). The MsFAMeT in silico analysis showed that greatest sequence similarity is found in Apis mellifera and other insects, while relatively less similarity is shown in crustaceans. Evidence of alternative splicing of a 27 nucleotide (nt) microexon explains the presence of the detected isoforms, 1 and 2. The expression analysis of the two isoforms showed a marked difference when castes were compared, suggesting that they could be involved differently in the JH metabolism in M. scutellaris, providing new insights for the comprehension of female plasticity.     

Changes in the content of phenolic compounds and IAA-oxidase activity during the growth of tobacco crown gall suspension culture

Phenolic content and IAA-oxidase (IAA-o) activity have been assayed in cells and medium of tobacco crown gall suspension culture in several stages of culture cycle. The highest content of total phenolics in the cells were found prior to cell division and in the middle stage of intensive growth. The beginning of intensive growth is accompanied by temporary reduction in phenolic level in the cells as well as their intensive secretion to the medium. In the second part of the culture cycle, when the phenolic production was weaker, the majority of these compounds were maintained in the cells. The highest activity of IAA-o in the cells was detected in the middle stage of intensive growth, simultaneously with high phenolic content; following it a considerable decrease of IAA-o activity is correlated with maximum of chlorogenic acid (ChA) content (at reduced amount of total phenolics). IAA-o activity increased again at the end of the stage of intensive growth when the level of phenolics was low including ChA. These data suggest that IAA-o in relation to phenolic level determines cell growth in the culture. In the culture medium — fairly distinct negative correlation between IAA-o activity and phenolic content suggests that the latter participates in enzyme activity regulation. During intensive growth IAA-o activity is strongly inhibited. The results prove that phenolic level. IAA-o activity and auxin level are closely correlated and may constitute essential elements of a mechanism of regulation crown gall cell growth in culture.     

Antisense inhibition of the sucrose transporter in potato: effects on amount and activity

The sucrose proton-cotransporter gene from potato (StSUT1) is mainly expressed in the phloem of mature, exporting leaves. To study the in vivo role of the protein, potato plants were transformed with antisense constructs of the sucrose transporter cDNA under control of the CaMV35S and the rolC promoters, respectively. Both types of transgenic plant develop symptoms characteristic of an inhibition of phloem loading. To determine the level of inhibition, immunological and transport studies were performed. Purified antibodies directed against a peptide from the central loop of SUT1 recognized a transporter with an apparent molecular mass of 47 kDa in leaf plasma membrane vesicles. Antisense repression under control of the non-specific CaMV35S promoter led to a strong reduction in SUT1 protein, whereas no such reduction could be detected when the companion cell-specific rolC promoter was used. Similarily. sucrose uptake in plasma membrane vesicles was reduced by 50–75% in CaMV35S but not in rolC plants. These data suggest that, unlike the rolC promoter, the sucrose transporter is expressed not only in the companion cells but also in other leaf cells. However, inhibition of the transporter by rolC-controlled antisense repression is sufficient to impair phloem loading.     

Identification and characterization of 3-ketosphinganine reductase activity encoded at the BT_0972 locus in Bacteroides thetaiotaomicron

Bacterial sphingolipid synthesis is important for the fitness of gut commensal bacteria with an implied potential for regulating mammalian host physiology. Multiple steps in bacterial sphingolipid synthesis pathways have been characterized previously, with the first step of de novo sphingolipid synthesis being well conserved between bacteria and eukaryotes. In mammals, the subsequent step of de novo sphingolipid synthesis is catalyzed by 3-ketosphinganine reductase, but the protein responsible for this activity in bacteria has remained elusive. In this study, we analyzed the 3-ketosphinganine reductase activity of several candidate proteins in Bacteroides thetaiotaomicron chosen based on sequence similarity to the yeast 3-ketosphinganine reductase gene. We further developed a metabolomics-based 3-ketosphinganine reductase activity assay, which revealed that a gene at the locus BT_0972 encodes a protein capable of converting 3-ketosphinganine to sphinganine. Taken together, these results provide greater insight into pathways for bacterial sphingolipid synthesis that can aid in future efforts to understand how microbial sphingolipid synthesis modulates host-microbe interactions.     

Exogenous caffeic acid inhibits the growth and enhances the lignification of the roots of soybean (Glycine max)

The allelopathic effect of caffeic acid was tested on root growth, phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, hydrogen peroxide (H₂O₂) accumulation, lignin content and monomeric composition of soybean (Glycine max) roots. We found that exogenously applied caffeic acid inhibited root growth, decreased the PAL activity and H₂O₂ content and increased the soluble and cell wall-bound POD activities. The p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monomers and total lignin (H + G + S) increased in the caffeic acid-exposed roots. When applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), caffeic acid equalized the inhibitory effect of PIP, whereas the application of methylene dioxocinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL) plus caffeic acid decreased lignin production. These results indicate that exogenously applied caffeic acid can be channeled into the phenylpropanoid pathway via the 4CL reaction, resulting in an increase of lignin monomers that solidify the cell wall and inhibit root growth.