Showing papers on "Phenylalanine published in 2010"

Branched-chain and aromatic amino acid catabolism into aroma volatiles in Cucumis melo L. fruit

Abstract: The unique aroma of melons (Cucumis melo L., Cucurbitaceae) is composed of many volatile compounds biosynthetically derived from fatty acids, carotenoids, amino acids, and terpenes. Although amino acids are known precursors of aroma compounds in the plant kingdom, the initial steps in the catabolism of amino acids into aroma volatiles have received little attention. Incubation of melon fruit cubes with amino acids and a-keto acids led to the enhanced formation of aroma compounds bearing the side chain of the exogenous amino or keto acid supplied. Moreover, L-[ 13 C6]phenylalanine was also incorporated into aromatic volatile compounds. Amino acid transaminase activities extracted from the flesh of mature melon fruits converted L-isoleucine, L-leucine, L-valine, L-methionine, or L-phenylalanine into their respective a-keto acids, utilizing a-ketoglutarate as the amine acceptor. Two novel genes were isolated and characterized (CmArAT1 and CmBCAT1) encoding 45.6 kDa and 42.7 kDa proteins, respectively, that displayed aromatic and branched-chain amino acid transaminase activities, respectively, when expressed in Escherichia coli. The expression of CmBCAT1 and CmArAT1 was low in vegetative tissues, but increased in flesh and rind tissues during fruit ripening. In addition, ripe fruits of climacteric aromatic cultivars generally showed high expression of CmBCAT1 and CmArAT1 in contrast to non-climacteric non-aromatic fruits. The results presented here indicate that in melon fruit tissues, the catabolism of amino acids into aroma volatiles can initiate through a transamination mechanism, rather than decarboxylation or direct aldehyde synthesis, as has been demonstrated in other plants.

High molecular size humic substances enhance phenylpropanoid metabolism in maize (Zea mays L.).

Abstract: A high molecular weight humic fraction (>3,500 Da) was characterized chemically by DRIFT and 1H NMR spectroscopy, and was applied to Zea mays L. plants to evaluate its effect on phenylpropanoid metabolism. The activity and gene expression of phenylalanine (tyrosine) ammonia-lyase (PAL/TAL), and the concentrations of phenolics and their amino acid precursors phenylalanine and tyrosine were assayed. Maximum induction of PAL/TAL activity and expression was obtained when the concentration of added humic substance was 1 mg C/l hydroponic solution. Phenylalanine and tyrosine significantly decreased (−16% and −22%, respectively), and phenolic compounds increased in treated plants. The effects of the humic substance could be ascribed partly to indoleacetic acid (27 nmol/mg C) in the humic fraction. Our results suggest that this humic fraction induces changes in phenylpropanoid metabolism. This is the first study that shows a relationship between humic substances and the phenylpropanoid pathway.

Oxidation of Amino Acids, Peptides and Proteins by Ozone: A Review

Abstract: The kinetics and products of the reaction of ozone with specific amino acids, peptides, and proteins are reviewed based on studies reported in the literature. Ozone reacts mainly with the unprotonated amino group of the acids and the second-order ozone rate constants for these reactions, except for cysteine, methionine, and tryptophan, vary by about two-orders from 2.6 × 104 to 4.4 × 106 M−1s−1. The site of attack on cysteine and methionine by O3 is at the sulfhydryl rather than the amino group to give sequential O-atom addition products. The order of reactivity for the oxidation of amino acids by O3 at pH 8 is cysteine > tryptophan ≈ methionine > phenylalanine ≈ histidine > others, with half-lives mostly in the range of milliseconds to tens of seconds (1 mg L-1 O3 dose). Reactions of O3 with aliphatic amino acids form nitrate, ammonia, and one or two carbon atom-containing carbonyl and carboxylic byproducts. In the ozonolysis of peptides and proteins, oxidation by O3 occurs at the tyrosine, tryptophan, h...

A mutation in amino acid permease AAP6 reduces the amino acid content of the Arabidopsis sieve elements but leaves aphid herbivores unaffected.

Abstract: The aim of this study was to investigate the role of the amino acid permease gene AAP6 in regulating phloem amino acid composition and then to determine the effects of this altered diet on aphid performance. A genotype of Arabidopsis thaliana (L.) was produced in which the function of the amino acid permease gene AAP6 (At5g49630) was abolished. Plants homozygous for the insertionally inactivated AAP6 gene had a significantly larger mean rosette width than the wild type and a greater number of cauline leaves. Seeds from the aap6 mutant were also significantly larger than those from the wild-type plants. Sieve element (SE) sap was collected by aphid stylectomy and the amino acids derivatized, separated, and quantified using Capillary Electrophoresis with Laser Induced Fluorescence (CE-LIF). In spite of the large variation across samples, the total amino acid concentration of SE sap of the aap6 mutant plants was significantly lower than that of the wild-type plants. The concentrations of lysine, phenylalanine, leucine, and aspartic acid were all significantly lower in concentration in the aap6 mutant plants compared with wild-type plants. This is the first direct demonstration of a physiological role for an amino acid transporter in regulating SE composition in vivo. The amino acid availability in sieve element sap is thought to be the major limiting factor for aphid growth and reproduction. Despite the changes in their diet, the aphid Myzus persicae (Sulzer) displayed only small changes in feeding behaviour on mutant plants when measured using the Electronic Penetration Graph (EPG) technique. Salivation by the aphid into the SE (E1 phase) was increased on mutant plants but there was no significant effect on other feeding EPG behaviours, or in the rate of honeydew production. Consistent with the small effect on aphid feeding behaviour, there was only a small effect of reduced sieve element amino acid concentration on aphid reproduction. The data are discussed in relation to the regulation of phloem composition and the role of phloem amino acids in regulating aphid performance.

Digestion rate of dietary starch affects systemic circulation of amino acids in weaned pigs.

Abstract: The present study was conducted to evaluate the in vitro and in vivo digestibility of dietary starch and its digestive behaviour on the systemic circulating amino acids (AA) in weaned pigs. Eighteen weanling pigs surgically fitted with a catheter in the jugular vein were randomly assigned to three dietary treatment groups. Sticky rice starch (SRS) was hydrolysed more quickly in vitro (P < 0.05) than maize starch (MS) and resistant starch (RS), and was almost completely hydrolysed within 4 h. The in vivo digestibility of dietary starch in different segments of the small intestine was significantly different. SRS was digested (81.9 %; P < 0.05) in the anterior jejunum, but not more than half of the MS and RS was digested in the same segment of the small intestine. The digestibilities of isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, aspartate and serine in the SRS group were higher than in the MS group (P < 0.05), and all nutritionally indispensable and dispensable AA in the SRS group were higher when compared with those in the RS group (P < 0.05). The serum concentrations of nutritionally indispensable AA, proline and serine in the three groups were increased to a peak point within 1.5 h postprandially then decreased gradually; however, the time that serum concentrations of alanine, aspartate, glutamate and glycine in each group increased to a peak point was different. The concentrations of nutritionally indispensable AA, including arginine, cystine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine at 09.30 hours and arginine, cystine, histidine, isoleucine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine at 13.30 hours in the SRS group were higher than in the MS group (P < 0.05); all nutritionally indispensable AA in the SRS group were higher than in the RS group at 09.30 and 13.30 hours (P < 0.05), respectively. We conclude that dietary starches digested rapidly in vitro have higher digestibility in the anterior small intestine of pigs. Diets containing rapidly digestible starch ameliorate the digestive and absorptive function and regulate AA metabolism to beneficially increase the entry of dietary AA into the systemic circulation in pigs.

Amino acid metabolism of bovine blastocysts: a biomarker of sex and viability.

Abstract: The ratio of male/female embryos may be modified by environmental factors such as maternal diet in vivo and the composition of embryo culture media in vitro. We have used amino acid profiling, a noninvasive marker of developmental potential to compare the effect of sex on the metabolism of bovine blastocysts conceived in vivo and in vitro. Blastocysts were incubated individually for 24 hr in a close-to-physiological mixture of amino acids and the depletion or appearance of 18 amino acids measured using HPLC. Blastocysts were then sexed by PCR. Amino acid depletion by in vitro-produced blastocysts and expanded blastocysts was higher than in embryos conceived in vivo (P = 0.02). When cultured in vitro, female embryos exhibited increased depletion of arginine, glutamate, and methionine and appearance of glycine, while male embryos displayed increased depletion of phenylalanine, tyrosine, and valine. Overall, in vitro-produced blastocysts exhibited sex-specific differences in metabolic profiles of 7 out of 18 amino acids; in vivo-produced, in 2 out of 18. These differences had disappeared by the expanded blastocyst stages. We have also shown that amino acid metabolism can predict the ability of bovine zygotes to develop to the blastocyst stage, providing “proof of principle” for the use of this technology in clinical IVF to select single embryos for transfer and thereby avoid the problem of multiple births. Mol. Reprod. Dev. 77: 285–296, 2010. © 2010 Wiley-Liss, Inc.

Determination of amino acids in Chinese rice wine by fourier transform near-infrared spectroscopy.

Abstract: Chinese rice wine is abundant in amino acids. The possibility of quantitative detection of 16 free amino acids (aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, and arginine) in Chinese rice wine by Fourier transform near-infrared (NIR) spectroscopy was investigated for the first time in this study. A total of 98 samples from vintage 2007 rice wines with different aging times were analyzed by NIR spectroscopy in transmission mode. Calibration models were developed using partial least-squares regression (PLSR) with high-performance liquid chromatography (HPLC) by postcolumn derivatization and diode array detection as a reference method. To validate the calibration models, full cross (leave-one-out) validation was employed. The results showed that the calibration statistics were good (rcal>0.94) for all amino acids except proline, histidine, and arginine. The correlation coefficient in cross validation (rcv) was >0.81 for 12 amino acids. The residual predictive deviation (RPD) value obtained was >1.5 in all amino acids except proline and arginine, and it was >2.0 in 6 amino acids. The results obtained in this study indicated that NIR spectroscopy could be used as an easy, rapid, and novel tool to quantitatively predict free amino acids in Chinese rice wine without sophisticated methods.

Antioxidant and angiotensin converting enzyme-inhibitory properties of a flaxseed protein-derived high Fischer ratio peptide mixture.

Abstract: Hydrolysis of flaxseed proteins using thermolysin and pronase followed by mixing with activated carbon, centrifugation and filtration yielded a filtrate (peptide mixture) with a Fischer ratio (branched-chain amino acids/aromatic amino acids) of 23.65 and a phenylalanine + tyrosine content of 1.11%. Gel permeation chromatography showed that the flaxseed peptide sample contained mainly low molecular weight peptides (<4 kDa). The high Fischer ratio peptide sample exhibited antioxidant property by scavenging 2,2-diphenyl-1-picrylhydrazyl radical, superoxide radical, hydroxyl radical, and also by protecting linoleic acid from oxidation. In addition, the peptide mixture showed potential antihypertensive properties by inhibiting angiotensin I-converting enzyme in a mixed-type inhibition pattern. Protein hydrolysates with Fischer ratio higher than 20 and phenylalanine + tyrosine content lower than 2% have been used to treat patients with hepatic encephalopathy; thus, this multifunctional flaxseed peptide mixture could be used to formulate food products with multiple human health benefits during liver diseases, oxidative stress and hypertension.

A Metabolomic Approach to Study Major Metabolite Changes during Acclimation to Limiting CO2 in Chlamydomonas reinhardtii

Abstract: Using a gas chromatography-mass spectrometry-time of flight technique, we determined major metabolite changes during induction of the carbon-concentrating mechanism in the unicellular green alga Chlamydomonas reinhardtii. In total, 128 metabolites with significant differences between high- and low-CO2-grown cells were detected, of which 82 were wholly or partially identified, including amino acids, lipids, and carbohydrates. In a 24-h time course experiment, we show that the amino acids serine and phenylalanine increase transiently while aspartate and glutamate decrease after transfer to low CO2. The biggest differences were typically observed 3 h after transfer to low-CO2 conditions. Therefore, we made a careful metabolomic examination at the 3-h time point, comparing low-CO2 treatment to high-CO2 control. Five metabolites involved in photorespiration, 11 amino acids, and one lipid were increased, while six amino acids and, interestingly, 21 lipids were significantly lower. Our conclusion is that the metabolic pattern during early induction of the carbon-concentrating mechanism fit a model where photorespiration is increasing.

Health consequences of catabolic synthesis of hippuric acid in humans.

Abstract: Hippuric acid has been a major human metabolite for years. However, there is no well-known documented health benefit associated with it except for excretion of environmental-toxic exposures of aromatic compounds such as toluene, or from dietary protein degradation and re-synthesis by intestinal microflora metabolism of quinic acid via the shikimate pathway. Thus hippuric acid can appear in humans as an excretory product from natural or unnatural sources. It has been believed over the years that the major source of urinary hippuric acid levels in humans has come from environmental toxic solvent exposures. However, more recently it was been shown that approximately 1-2 mM hippuric acid is excreted daily in the urine, even in the absence of organic solvent exposure, signalling abundant metabolic dietary sources of hippuric acid are also apparent. One of these has been dietary proteins. The other is from the well-documented presence of quinic acid in healthy colored foodstuffs. Quinic acid is a key metabolite associated with the shikimate pathway existing only in plants, and it is responsible for essential amino acid biosynthesis such as tryptophan, phenylalanine and tyrosine. Here we review the evidence that the human gastrointestinal tract microflora are responsible for quinic acid metabolism not only to hippuric acid, but more importantly to efficacious antioxidant amino acids and vitamins.

Lipid peroxyl radicals mediate tyrosine dimerization and nitration in membranes

Abstract: Protein tyrosine dimerization and nitration by biologically relevant oxidants usually depend on the intermediate formation of tyrosyl radical ((*)Tyr) In the case of tyrosine oxidation in proteins associated with hydrophobic biocompartments, the participation of unsaturated fatty acids in the process must be considered since they typically constitute preferential targets for the initial oxidative attack Thus, we postulate that lipid-derived radicals mediate the one-electron oxidation of tyrosine to (*)Tyr, which can afterward react with another (*)Tyr or with nitrogen dioxide ((*)NO(2)) to yield 3,3'-dityrosine or 3-nitrotyrosine within the hydrophobic structure, respectively To test this hypothesis, we have studied tyrosine oxidation in saturated and unsaturated fatty acid-containing phosphatidylcholine (PC) liposomes with an incorporated hydrophobic tyrosine analogue BTBE (N-t-BOC l-tyrosine tert-butyl ester) and its relationship with lipid peroxidation promoted by three oxidation systems, namely, peroxynitrite, hemin, and 2,2'-azobis (2-amidinopropane) hydrochloride In all cases, significant tyrosine (BTBE) oxidation was seen in unsaturated PC liposomes, in a way that was largely decreased at low oxygen concentrations Tyrosine oxidation levels paralleled those of lipid peroxidation (ie, malondialdehyde and lipid hydroperoxides), lipid-derived radicals and BTBE phenoxyl radicals were simultaneously detected by electron spin resonance spin trapping, supporting an association between the two processes Indeed, alpha-tocopherol, a known reactant with lipid peroxyl radicals (LOO(*)), inhibited both tyrosine oxidation and lipid peroxidation induced by all three oxidation systems Moreover, oxidant-stimulated liposomal oxygen consumption was dose dependently inhibited by BTBE but not by its phenylalanine analogue, BPBE (N-t-BOC l-phenylalanine tert-butyl ester), providing direct evidence for the reaction between LOO(*) and the phenol moiety in BTBE, with an estimated second-order rate constant of 48 x 10(3) M(-1) s(-1) In summary, the data presented herein demonstrate that LOO(*) mediates tyrosine oxidation processes in hydrophobic biocompartments and provide a new mechanistic insight to understand protein oxidation and nitration in lipoproteins and biomembranes

Stimulation of Nonselective Amino Acid Export by Glutamine Dumper Proteins

Abstract: Phloem and xylem transport of amino acids involves two steps: export from one cell type to the apoplasm, and subsequent import into adjacent cells. High-affinity import is mediated by proton/amino acid cotransporters, while the mechanism of export remains unclear. Enhanced expression of the plant-specific type I membrane protein Glutamine Dumper1 (GDU1) has previously been shown to induce the secretion of glutamine from hydathodes and increased amino acid content in leaf apoplasm and xylem sap. In this work, tolerance to low concentrations of amino acids and transport analyses using radiolabeled amino acids demonstrate that net amino acid uptake is reduced in the glutamine-secreting GDU1 overexpressor gdu1-1D. The net uptake rate of phenylalanine decreased over time, and amino acid net efflux was increased in gdu1-1D compared with the wild type, indicating increased amino acid export from cells. Independence of the export from proton gradients and ATP suggests that overexpression of GDU1 affects a passive export system. Each of the seven Arabidopsis (Arabidopsis thaliana) GDU genes led to similar phenotypes, including increased efflux of a wide spectrum of amino acids. Differences in expression profiles and functional properties suggested that the GDU genes fulfill different roles in roots, vasculature, and reproductive organs. Taken together, the GDUs appear to stimulate amino acid export by activating nonselective amino acid facilitators.

Phenylketonuria: a 21st century perspective.

Abstract: Phenylketonuria is the most prevalent inherited defect in amino acid metabolism. Owing to mutations in the gene encoding the enzyme phenylalanine hydroxylase, the essential amino acid phenylalanine cannot be hydroxylated to tyrosine and blood and tissue concentrations of phenylalanine increase. Untreated, phenylketonuria causes severe mental retardation, epilepsy and behavioral problems. The combined effect of neonatal screening and treatment has, however, meant that phenylketonuria is now a biochemical rather than a clinical diagnosis. Treatment consists of stringent dietary restriction of natural protein intake and supplementation of amino acids other than phenylalanine by a chemically manufactured protein substitute. Although clinical outcome on a phenylalanine-restricted diet is good, neuropsychological deficits are now known to exist in dietary-treated patients with phenylketonuria, and quality of life, nutritional condition and psychosocial outcome could probably also be improved. The need for new therapeutic approaches is being met by supplementation with tetrahydrobiopterin or large neutral amino acids, whilst development of the use of phenylalanine ammonia lyase, and, in the longer term, gene therapy and chaperone treatment holds promise. This Review provides an overview of the history of phenylketonuria, the challenges of treatment today and the treatment possibilities in the near future.

Pleiotropic physiological consequences of feedback-insensitive phenylalanine biosynthesis in Arabidopsis thaliana.

Abstract: A large proportion of plant carbon flow passes through the shikimate pathway to phenylalanine, which serves as a precursor for numerous secondary metabolites. To identify new regulatory mechanisms affecting phenylalanine metabolism, we isolated Arabidopsis thaliana mutants that are resistant to the phytotoxic amino acid m-tyrosine, a structural analog of phenylalanine. Map-based cloning identified adt2-1D, a dominant point mutation causing a predicted serine to alanine change in the regulatory domain of ADT2 (arogenate dehydratase 2). Relaxed feedback inhibition and increased expression of the mutant enzyme caused up to 160-fold higher accumulation of free phenylalanine in rosette leaves, as well as altered accumulation of several other primary and secondary metabolites. In particular, abundance of 2-phenylethylglucosinolate, which is normally almost undetectable in leaves of the A. thaliana Columbia-0 accession, is increased more than 30-fold. Other observed phenotypes of the adt2-1D mutant include abnormal leaf development, resistance to 5-methyltryptophan, reduced growth of the generalist lepidopteran herbivore Trichoplusia ni (cabbage looper) and increased salt tolerance.

Influence of nitrogen on the primary and secondary metabolism and synthesis of flavonoids in Chrysanthemum morifolium Ramat.

Abstract: The effects of nitrogen (N) supply on nitrogen metabolism in leaves of Chrysanthemum morifolium Ramat. were examined in five different stages throughout the growing season. The results suggested that flavonoids content was positively related to phenylalanine ammonia-lyase activity through the whole growing stage of the plant but with a decreasing correlation coefficient for increasing nitrogen supply. There was no correlation between flavonoids and 4-coumarate coenzyme A ligase. Soluble protein content was positively correlated with phenylalanine ammonia-lyase activity because there was little competition for the phenylalanine in the leaves under low nitrogen supply. Phenylalanine ammonia-lyase activity decreased gradually with increasing nitrogen supply because of the competition for the phenylalanine in protein synthesizes. The results suggest that nitrogen nutrition plays a key role in biosynthesis of enzymes in the leaves of C. morifolium.

Partitioning of New Carbon as 11C in Nicotiana tabacum Reveals Insight into Methyl Jasmonate Induced Changes in Metabolism

Abstract: We examined the timeline by which methyl jasmonate (MeJA) reprograms new carbon partitioning into key metabolite pools. The radioactive isotope 11C (t1/2 20.4 min), administered to intact leaves of Nicotiana tabacum L. (cv Samsun) as 11CO2 gas enabled us to measure changes in new carbon partitioning into soluble sugar and amino acid pools of [11C]photosynthate. A 500 μM MeJA treatment resulted in a decrease in the [11C]soluble sugar pool and an increase in the [11C]amino acid pool after 4 h. This pattern was more pronounced 15 h after treatment. We also examined the timeline for 11C-partitioning into aromatic amino acid metabolites of the shikimate pathway. [11C]Tyrosine, [11C]phenylalanine and [11C]tryptophan were elevated 1.5-fold, 12-fold and 12-fold, respectively, relative to controls, 4 h after MeJA treatment, while endogeneous pools were unchanged. This suggests that only new carbon is utilized during early stages of defense induction. By 15 h, [11C]tyrosine and [11C]phenylalanine returned to baseline while [11C]tryptophan was elevated 30-fold, suggesting that MeJA exerts selective control over the shikimate pathway. Finally, we measured trans-cinnamic acid levels as a gauge of downstream phenolic metabolism. Levels were unchanged 4 h after MeJA treatment relative to controls, but were increased 2-fold by 15 h, indicating a lag in response of secondary metabolism.

Mutational analysis of phenylalanine ammonia lyase to improve reactions rates for various substrates.

Abstract: Phenylalanine ammonia lyases (PAL) catalyze the reversible, non-reductive amination of trans-cinnamic acid to l-phenylalanine in the presence of high ammonia concentrations. Since neither cofactor recycling nor other additives are needed and by this asymmetric synthesis theoretical yields of 100% can be reached, it is an interesting reaction for industrial processes. In this study we demonstrate the superior properties of p-nitro-cinnamic acid (p-n-CA) in the amination reaction using the PAL from Petroselinum crispum (pcPAL). By focused-directed evolution, three mutants were identified showing increased reaction rates and decreased substrate inhibition. Together, the F137V mutant with p-n-CA showed a 15-fold increased reaction rate compared with the pcPAL WT with the natural cinnamic acid. The high reaction rates were also proven in preparative scale experiments. Activities towards other p-substituted cinnamic acids showing different electronic effects of the substituent were analyzed. Focused-directed evolution around the carboxylic acid- and amine-binding site always decreased PAL activity, due to a sensitive H-bond network.

Comparison of adeno-associated virus pseudotype 1, 2, and 8 vectors administered by intramuscular injection in the treatment of murine phenylketonuria.

Abstract: Phenylketonuria (PKU) is caused by hepatic phenylalanine hydroxylase (PAH) deficiency and is associated with systemic accumulation of phenylalanine (Phe). Previously we demonstrated correction of murine PKU after intravenous injection of a recombinant type 2 adeno-associated viral vector pseudotyped with type 8 capsid (rAAV2/8), which successfully directed hepatic transduction and Pah gene expression. Here, we report that liver PAH activity and phenylalanine clearance were also restored in PAH-deficient mice after simple intramuscular injection of either AAV2 pseudotype 1 (rAAV2/1) or rAAV2/8 vectors. Serotype 2 AAV vector (rAAV2/2) was also investigated, but long-term phenylalanine clearance has been observed only for pseudotypes 1 and 8. Therapeutic correction was shown in both male and female mice, albeit more effectively in males, in which correction lasted for the entire period of the experiment (>1 year). Although phenylalanine levels began to rise in female mice at about 8-10 months after rAAV2/8 injection they remained only mildly hyperphenylalaninemic thereafter and subsequent supplementation with synthetic tetrahydrobiopterin resulted in a transient decrease in blood phenylalanine. Alternatively, subsequent administration of a second vector with a different AAV pseudotype to avoid immunity against the previously administrated vector was also successful for long-term treatment of female PKU mice. Overall, this relatively less invasive gene transfer approach completes our previous studies and allows comparison of complementary strategies in the development of efficient PKU gene therapy protocols.

Expression of bacterial tyrosine ammonia-lyase creates a novel p-coumaric acid pathway in the biosynthesis of phenylpropanoids in Arabidopsis

Abstract: Some flavonoids are considered as beneficial compounds because they exhibit anticancer or antioxidant activity. In higher plants, flavonoids are secondary metabolites that are derived from phenylpropanoid biosynthetic pathway. A large number of phenylpropanoids are generated from p-coumaric acid, which is a derivative of the primary metabolite, phenylalanine. The first two steps in the phenylpropanoid biosynthetic pathway are catalyzed by phenylalanine ammonia-lyase and cinnamate 4-hydroxylase, and the coupling of these two enzymes forms a rate-limiting step in the pathway. For the generation of p-coumaric acid, the conversion from phenylalanine to p-coumaric acid that is catalyzed by two enzymes can be theoretically performed by a single enzyme, tyrosine ammonia-lyase (TAL) that catalyzes the conversion of tyrosine to p-coumaric acid in certain bacteria. To modify the p-coumaric acid pathway in plants, we isolated a gene encoding TAL from a photosynthetic bacterium, Rhodobacter sphaeroides, and introduced the gene (RsTAL) in Arabidopsis thaliana. Analysis of metabolites revealed that the ectopic over-expression of RsTAL leads to higher accumulation of anthocyanins in transgenic 5-day-old seedlings. On the other hand, 21-day-old seedlings of plants expressing RsTAL showed accumulation of higher amount of quercetin glycosides, sinapoyl and p-coumaroyl derivatives than control. These results indicate that ectopic expression of the RsTAL gene in Arabidopsis enhanced the metabolic flux into the phenylpropanoid pathway and resulted in increased accumulation of flavonoids and phenylpropanoids.

First Hyperpolarizability of the Natural Aromatic Amino Acids Tryptophan, Tyrosine, and Phenylalanine and the Tripeptide Lysine−Tryptophan−Lysine Determined by Hyper-Rayleigh Scattering

Abstract: We report the first hyperpolarizability of tryptophan (Trp) and tyrosine (Tyr) and an upper limit for that of phenylalanine (Phe), three natural aromatic amino acids. The measurements were performed with hyper-Rayleigh scattering in an aqueous Tris buffer solution at a pH of 8.5 and 150 mM salt concentration with a fundamental wavelength of 780 nm. A value of (4.7 ± 0.7) × 10−30 esu is found for Trp and (4.1 ± 0.7) × 10−30 esu for Tyr whereas the upper limit of 1.4 × 10−30 esu is found for that of Phe due to its limited solubility. The influence of the presence of lysine (Lys) in close vicinity of Trp is investigated with a measurement of the first hyperpolarizabilty of Trp in an excess of Lys and compared to the first hyperpolarizability obtained for the tripeptide Lys-Trp-Lys. The clear decrease of the values measured in these two cases indicates that the first hyperpolarizabilty of Trp is very sensitive to its local environment.

Fibroblast growth factor mutants having improved functional half-life and methods of their use

Abstract: Mutant fibroblast growth factor (FGF) proteins having a polypeptide sequence with a high sequence identity to proteins encoded by members of the Fgf-1 subfamily of genes from a mammalian species, such as human, and with a specific amino acid substitution of an alanine at a position corresponding to amino acid position 66 of human FGF-1 with a cysteine and/or a specific amino acid substitution of a phenylalanine at a position corresponding to amino acid position 132 of human FGF-1 with a tryptophan (based on the 140 amino acid numbering scheme of human FGF-1) are provided. Other amino acid mutations or substitutions may be combined. Polynucleotide sequences encoding the mutant FGF proteins and host cells containing such polynucleotide sequences are provided. Methods of administering a mutant FGF protein to an individual to treat an ischemic condition or disease or a wound or tissue injury are also provided.

Regulation of phenylalanine hydroxylase: conformational changes upon phenylalanine binding detected by hydrogen/deuterium exchange and mass spectrometry.

Abstract: Phenylalanine acts as an allosteric activator of the tetrahydropterin-dependent enzyme phenylalanine hydroxylase. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into local conformational changes accompanying activation of rat phenylalanine hydroxylase by phenylalanine. Peptides in the regulatory and catalytic domains that lie in the interface between these two domains show large increases in the extent of deuterium incorporation from solvent in the presence of phenylalanine. In contrast, the effects of phenylalanine on the exchange kinetics of a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site for the amino acid substrate. These results support a model in which the N-terminus of the protein acts as an inhibitory peptide, with phenylalanine binding causing a conformational change in the regulatory domain that alters the interaction between the catalytic and regulatory domains.

Identification and characterization of the PhhR regulon in Pseudomonas putida

Abstract: Pseudomonas putida is a soil microorganism that utilizes aromatic amino acids present in root exudates as a nitrogen source. We have previously shown that the PhhR transcriptional regulator induces phhAB genes encoding a phenylalanine hydroxylase. In this study we show, using microarray assays and promoter fusions, that PhhR is a global regulator responsible for the activation of genes essential for phenylalanine degradation, phenylalanine homeostasis and other genes of unknown function. Recently, it has been shown that phenylalanine catabolism occurs through more than one pathway. One of these possible pathways involves the metabolism of phenylalanine via tyrosine, p-hydroxyphenylpyruvate, and homogentisate. We identified two genes within this pathway that encode an acyl-CoA transferase involved in the metabolism of acetoacetate. All genes in this pathway were induced in response to phenylalanine in a PhhR-proficient background. The second potential degradative pathway involves the degradation of phenylalanine to produce phenylpyruvate, which seems to be degraded via phenylacetyl-CoA. A number of mutants in the paa genes encoding phenylacetyl-CoA degradation enzymes fail to grow on phenylpyruvate or phenylacetate, further supporting the existence of this second pathway. We found that the PhhR regulon also includes genes involved in the biosynthesis of aromatic amino acids that are repressed in the presence of phenylalanine, suggesting the possibility of feedback at the transcriptional level. In addition, we found that PhhR modulates the level of expression of the broad-substrate-specificity MexEF/OprN efflux pump. Expression from this pump is under the control of mexT gene product because phenylalanine-dependent transcription from the mexE promoter does not occur in a mexT mutant background. These results place PhhR as an important regulator in the control of bacterial responses to aromatic amino acids.

Characterisation of Amino Acid Modified Cellulose Surfaces using ToF-SIMS and XPS.

Abstract: Cellulosic fibrous networks are modified using 3 different amino acids; small (Glycine, Gly), aliphatic (Leucine, Leu) and aromatic (Phenylalanine, Phe). The effect of amino acid functionality on chemical coupling to cellulose fibres in terms of their coverage and packing density are investigated. Different amino acid modified cellulose networks are characterised by using Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and X-ray Photoelectron Spectroscopy (XPS). The presence of amino acids is confirmed using ToF-SIMS. The quantitative distribution of different amino acids across the cellulose surface is assessed by using XPS. It is shown that the packing density of amino acids depends on the size of the side chain; smaller amino acids (Gly, Leu) tend to couple to the surface at higher density compared to larger ones (Phe). This study has implications for the functionalisation of polysaccharide materials for a wide range of applications.

Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations

Abstract: Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using α-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 A resolution. The crystal structure revealed the interaction between the pyridoxal-5′-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.