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Ammonia Lyase (ammonia + lyase)
Kinds of Ammonia Lyase Selected AbstractsAlteration of the Diastereoselectivity of 3-Methylaspartate Ammonia Lyase by Using Structure-Based MutagenesisCHEMBIOCHEM, Issue 13 2009Hans Raj Abstract 3-Methylaspartate ammonia-lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)-3-methylaspartic acid and (2S,3R)-3-methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3 proton of the respective stereoisomer to generate an enolate anion intermediate that is stabilized by coordination to the essential active-site MgII ion. The crystal structure of MAL in complex with (2S,3S)-3-methylaspartic acid suggests that Lys331 is the only candidate in the vicinity that can function as a general base catalyst. The structure of the complex further suggests that two other residues, His194 and Gln329, are responsible for binding the C4 carboxylate group of (2S,3S)-3-methylaspartic acid, and hence are likely candidates to assist the MgII ion in stabilizing the enolate anion intermediate. In this study, the importance of Lys331, His194, and Gln329 for the activity and stereoselectivity of MAL was investigated by site-directed mutagenesis. His194 and Gln329 were replaced with either an alanine or arginine, whereas Lys331 was mutated to a glycine, alanine, glutamine, arginine, or histidine. The properties of the mutant proteins were investigated by circular dichroism (CD) spectroscopy, kinetic analysis, and 1H NMR spectroscopy. The CD spectra of all mutants were comparable to that of wild-type MAL, and this indicates that these mutations did not result in any major conformational changes. Kinetic studies demonstrated that the mutations have a profound effect on the values of kcat and kcat/KM; this implicates Lys331, His194 and Gln329 as mechanistically important. The 1H NMR spectra of the amination and deamination reactions catalyzed by the mutant enzymes K331A, H194A, and Q329A showed that these mutants have strongly enhanced diastereoselectivities. In the amination direction, they catalyze the conversion of mesaconate to yield only (2S,3S)-3-methylaspartic acid, with no detectable formation of (2S,3R)-3-methylaspartic acid. The results are discussed in terms of a mechanism in which Lys331, His194, and Gln329 are involved in positioning the substrate and in formation and stabilization of the enolate anion intermediate. [source] Inhibition of Histidine Ammonia Lyase by Heteroaryl-alanines and AcrylatesCHEMISTRY & BIODIVERSITY, Issue 5 2006Adrian Katona Abstract Histidine ammonia lyase (HAL) catalyzes the elimination of ammonia from the substrate to form (E)-urocanate. The interaction between HAL and acrylic acids or alanines substituted with heteroaryl groups in the , -position was investigated. These proved to be strong competitive inhibitors when the heteroaryl groups were furanyl, thiophenyl, benzofuranyl, and benzothiophenyl, carrying the alanyl or acrylic side chains either in 2 or 3 positions, with Ki values between 18 and 139,,M. The exception was (furan-3-yl)alanine which was found to be inert. Tryptophan and 1-methyltryptophan, as well as the corresponding acrylates (=prop-2-enoates), are strong mixed inhibitors of HAL. Theoretically, L -histidine can be dissected into 4-methyl-1H -imidazole and glycine. Whereas these two compounds separately are only very weak inhibitors of HAL, equimolar amounts of both show a Ki value of 1.7±0.09,mM which is to be compared with the Km value of 15.6,mM for the normal reaction. We conclude that 5-methyl-1H -imidazole and glycine mimic the substrate and occupy the active site of HAL in a similar orientation. [source] Two beta-alanyl-CoA:ammonia lyases in Clostridium propionicumFEBS JOURNAL, Issue 3 2005Gloria Herrmann The fermentation of ,-alanine by Clostridium propionicum proceeds via activation to the CoA-thiol ester, followed by deamination to acryloyl-CoA, which is also an intermediate in the fermentation of l -alanine. By shifting the organism from the carbon and energy source ,-alanine to ,-alanine, the enzyme ,-alanyl-CoA:ammonia lyase is induced 300-fold (, 30% of the soluble protein). The low basal lyase activity is encoded by the acl1 gene, whereas the almost identical acl2 gene (six amino acid substitutions) is responsible for the high activity after growth on ,-alanine. The deduced ,-alanyl-CoA:ammonia lyase proteins are related to putative ,-aminobutyryl-CoA ammonia lyases involved in lysine fermentation and found in the genomes of several anaerobic bacteria. ,-Alanyl-CoA:ammonia lyase 2 was purified to homogeneity and characterized as a heteropentamer composed of 16 kDa subunits. The apparent Km value for acryloyl-CoA was measured as 23 ± 4 µm, independent of the concentration of the second substrate ammonia; kcat/Km was calculated as 107 m,1·s,1. The apparent Km for ammonia was much higher, 70 ± 5 mm at 150 µm acryloyl-CoA with a much lower kcat/Km of 4 × 103 m,1·s,1. In the reverse reaction, a Km of 210 ± 30 µM was obtained for ,-alanyl-CoA. The elimination of ammonia was inhibited by 70% at 100 mm ammonium chloride. The content of ,-alanyl-CoA:ammonia lyase in ,-alanine grown cells is about 100 times higher than that required to sustain the growth rate of the organism. It is therefore suggested that the enzyme is needed to bind acryloyl-CoA, in order to keep the toxic free form at a very low level. A formula was derived for the calculation of isomerization equilibra between l -alanine/,-alanine or d -lactate/3-hydroxypropionate. [source] Analysing scots pine defence-related transcripts and fungal DNA levels in seedlings single- or dual-inoculated with endophytic and pathogenic Rhizoctonia speciesFOREST PATHOLOGY, Issue 6 2009H. Grönberg Summary Fungal DNA and induction of host defence-related transcripts were monitored by real-time PCR in young Scots pine seedlings inoculated with pathogenic uninucleate (UNR) and endophytic binucleate (BNR) Rhizoctonia species. The UNR (teleomorph Ceratobasidium bicorne) causes root dieback in conifer seedlings following invasion of the vascular cylinder via root apex and destroying apical meristems whilst the BNR, representing anastomosis group AG-I of genus Ceratobasidium, is primarily restricted to the cortex in basal root regions. In the experiment 1 the fungi were simultaneously inoculated on roots, while in experiment 2, BNR was pre-inoculated 168 h before inoculation with UNR. Nucleic acids were extracted from infected roots at intervals up to 192 h post-infection (hpi), and the genomic DNA levels of the host and fungi and the transcript levels of a house-keeping gene (glyceraldehyde-3-phosphate dehydrogenase) and nine putative defence genes were quantified. In simultaneous inoculation UNR was more competitive than BNR whereas pre-inoculation of BNR suppressed but did not completely prevent root colonization by UNR. Stilbene synthase (STS) transcription was significantly up-regulated in single-inoculations with both fungi and in dual inoculation in both experiments. Maximum STS transcript levels were observed in roots single-inoculated with UNR; the peak level at 48 hpi in experiment 2 was significantly higher than in seedlings single-inoculated with BNR or co-inoculated with both fungi, the latter two treatments showing relatively similar STS transcript levels. Similarly, transcript levels of phenylalanine ammonia lyase at 48 hpi in experiment 2 were significantly higher in roots single-inoculated with UNR compared with BNR or in UNR+BNR co-inoculations. The other seven putative defence genes monitored did not show any clear-cut up-regulation following fungal inoculation. We conclude that BNR suppresses UNR in Scots pine roots via direct competition for infection sites, since the studied transcripts showed no evidence of BNR induced resistance against UNR. [source] Effects of heat treatment on the quality of fresh-cut Chinese water chestnutINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 2 2004Litao Peng Summary A heat treatment to inhibit browning and maintain the quality of fresh-cut Chinese water chestnut was developed. Slices of Chinese water chestnut, cv. Guilin, were immersed in boiling water for 30 s, placed into film-wrapped trays and then stored at 4 °C for up to 12 days. Changes in browning, eating quality and disease incidence were measured. The effect of heat treatment on the content of total phenolics and activities of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD) was also evaluated. The heat treatment effectively prevented browning associated with PAL, PPO and POD activities and total phenolic content and delayed the decrease in eating quality, which is associated with reduced total soluble solids, titratable acidity and ascorbic acid, compared with fresh-cut Chinese water chestnut. Inhibition of browning by heat treatment without microbial growth was achieved for 12 days of storage at 4 °C. These results showed that heat treatment effectively maintained the quality of fresh-cut Chinese water chestnut. [source] Efficient Tandem Biocatalytic Process for the Kinetic Resolution of Aromatic ,-Amino AcidsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 9 2010Bian Wu Abstract We describe a simple and efficient enzymatic tandem reaction for the preparation of enantiomerically pure , -phenylalanine and its analogues from the corresponding racemates. In this process, phenylalanine aminomutase (PAM) catalyzes the stereoselective isomerization of (R)- , -phenylalanines to (S)- , -phenylalanines, which are in situ transformed to cinnamic acids by phenylalanine ammonia lyase (PAL). Preparative scale conversions are done with a mutated PAM with enhanced catalytic activity. [source] Plant growth promotion and induction of resistance in Camellia sinensis by Bacillus megateriumJOURNAL OF BASIC MICROBIOLOGY, Issue 3 2006Usha Chakraborty Dr. Bacillus megaterium de Bary TRS-4 was isolated from tea rhizosphere and tested for its ability to promote growth and cause disease reduction in tea plants. In vivo studies revealed the ability of this bacterium to promote growth of tea plants very significantly. Brown root rot disease, caused by Fomes lamaoensis was markedly reduced by application of the bacterium to the soil. Population of F. lamaoensis in soil before and after application of B. megaterium, as determined by ELISA and dot-blot using PAb raised against the pathogen, was shown to be greatly reduced in presence of the bacterium. Biochemical changes induced in tea plants were also examined. Root colonization by B. megaterium and subsequent inoculation with F. lamaoensis also led to an increase in polyphenolics, as well as in defense related enzymes-peroxidase, chitinase, , -1,3-glucanase and phenyl alanine ammonia lyase. Determination of mechanism of action of this bacterium revealed it to be able to solubilize phosphate, produce IAA, siderophore and antifungal metabolite. The plant growth promotion and reduction of disease intensity have been shown to be due to a combination of several mechanisms. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] EFFECT OF OXYGEN CONCENTRATION ON THE BIOCHEMICAL AND CHEMICAL CHANGES OF STORED LONGAN FRUITJOURNAL OF FOOD QUALITY, Issue 1 2009G. CHENG ABSTRACT Longan fruits were stored for 6 days in atmosphere of 5, 21 (air) or 60% O2 (balance N2) at 28C and 90,95% relative humidity to examine effects of low and high O2 concentration on enzymatic browning and quality attributes of the fruit. Changes in pericarp browning, pulp breakdown, disease development, total phenol content, activities of phenol metabolism-associated enzymes, relative leakage rate, ,,, -diphenyl- , -picrylhydrazy (DPPH) radical scavenging activity, and contents of total soluble solids, titratable acidity and ascorbic acid were evaluated. Storage of fruit in a 5% O2 atmosphere markedly delayed pericarp browning in association with maintenance of high total phenolic content and reduced activities of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase. Moreover, the fruit stored in a 5% O2 atmosphere exhibited a lower relative leakage rate and higher DPPH radical scavenging activity than fruit stored in air. This presumably was beneficial in maintaining compartmentation of enzymes and substrates, and thus, reducing pericarp browning. Pulp breakdown and disease development were also reduced by exposure to a 5% oxygenatmosphere. On the contrary, exposure of longan fruit to a 60% O2 atmosphere accelerated pericarp browning, pulp breakdown and decay development. PPO and POD activities and relative leakage rate were similar for control and 60% O2 -treated fruit after 4 and 6 days of storage. Furthermore, treatment with 60% O2 significantly decreased the phenolic content and DPPH scavenging activity of fruit. In addition, exposure to 5 or 60% O2 resulted in a higher level of total soluble solids, but a lower level of ascorbic acid of longan fruit flesh. In conclusion, exposure to a 5% O2 atmosphere showed great potential to reduce pericarp browning and extend shelf life of longan fruit. PRACTICAL APPLICATIONS Pericarp browning and pulp breakdown are the major causes of deterioration in postharvest longan. Conventional controlled atmosphere with low O2 and high CO2 is effective in maintaining quality and extending shelf life of fruits and vegetables, including inhibition of tissue browning. In this study, 5%-controlled atmosphere reduced significantly pericarp browning, pulp breakdown and rot development. It could potentially be useful as a postharvest technology of longan fruit for reducing or replacing the use of chemicals such as SO2 and fungicides, but it requires further investigation. [source] Sanitation Procedure Affects Biochemical and Nutritional Changes of Shredded CarrotsJOURNAL OF FOOD SCIENCE, Issue 2 2007Saúl Ruiz-Cruz ABSTRACT:, Fresh-cut vegetables are considered convenient but with less nutritional quality compared to raw natural produce. Carrots are highly appreciated because of their carotene and antioxidant nutrients, but processing requires an appropriate sanitation procedure that ensures microbiological safety to consumers. The effect of the sanitation processing on the nutritional composition of shredded carrots was studied. Treatments tested were tap water, 200 ppm sodium hypochlorite (Cl), 40 ppm peroxyacetic acid (PA), and 100, 250, and 500 ppm acidified sodium chlorite (ASC). Measured parameters were oxygen radical absorbing capacity (ORAC), total phenolics and carotenoids, sugars, and phenylalanine ammonia lyase (PAL) and peroxidase (POD) activity. Shredded carrots sanitized with ASC retained higher levels of sugars, carotene, and antioxidant capacity. ASC also delayed the PAL and POD activity. These results show the importance of evaluating nutritional parameters during processing stages, since minimal processing does not necessarily imply loss of nutritional value. Furthermore, the availability of fresh-cut produce may increase the intake of nutrients, with a positive effect on health. [source] Synthesis of carbon-14 labeled [1- 14C]-, and [2- 14C]- L -tyrosineJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 8 2001W. Augustyniak Abstract Two labeled isotopomers of L -tyrosine, L -Tyr, have been synthesized using specific properties of the enzymes phenylanine ammonia lyase, PAL, and L -phenylalanine hydroxylase. In an intermediate step [1- 14C]-, and [2- 14C]- L -phenylalanine, L -Phe, have been obtained from [1- 14C]-, and [2- 14C]cinnamic acid, prepared from potassium [14C]cyanate or [2- 14C]malonic acid, and by addition of ammonia in the presence of enzyme PAL. The labeled isotopomers of L -Phe have been converted into [1- 14C]-, and [2- 14C]- L -Tyr using the enzyme L -phenylalanine hydroxylase. Copyright © 2001 John Wiley & Sons, Ltd. [source] Induction of Systemic Acquired Resistance in Arachis hypogaea L. by Sclerotium rolfsii Derived ElicitorsJOURNAL OF PHYTOPATHOLOGY, Issue 9 2010Durgesh Nandini Abstract Plants evolve a strategy to survive the attacks of potential pathogens by inducing the microbial signal molecules. In this study, plant defence responses were induced in four different varieties of Arachis hypogaea (J-11, GG-20, TG-26 and TPG41) using the fungal components of Sclerotium rolfsii in the form of fungal culture filtrate (FCF) and mycelial cell wall (MCW), and the levels of defence-related signal molecule salicylic acid (SA), marker enzymes such as peroxidase (POX), phenylalanine ammonia lyase (PAL), ,-1,3-glucanase and lignin were determined. There was a substantial fold increase in POX, PAL, SA, ,-1,3-glucanase and lignin content in FCF- and MCW-treated plants of all varieties of groundnut when compared to that of control plants. The enzyme activities were much higher in FCF-treated plants than in MCW-treated plants. The increase in fold activity of enzymes and signal molecule varied between different varieties. These results indicate that the use of fungal components (FCF and MCW) had successfully induced systemic resistance in the four different varieties of groundnut plants against Sclerotium rolfsii. [source] Cloning, Expression and Characterization of Protein Elicitors from the Soyabean Pathogenic Fungus Phytophthora sojaeJOURNAL OF PHYTOPATHOLOGY, Issue 3 2000J. Becker The oomycete Phytophthora sojae is a severe pathogen of soybean. Several resistance genes against races of P. sojae exist in soybean but the nature of corresponding avirulence genes is unknown. Clones encoding four different isoforms of a protein elicitor from P. sojae (sojein 1,4) belonging to the class of acidic ,-elicitins have been isolated. These 98 amino acid proteins show high homology to elicitins from other Phytophthora species. The different sojein isoforms were expressed in Escherichia coli as His-tagged fusion proteins. Purified sojein as well as recombinant sojein isoforms induce hypersensitive reaction (HR)-like lesions in tobacco but are not active as race-specific elicitors in soybean. However all sojein isoforms induce defence-related genes like those encoding phenylalanine ammonia lyase, glutathione-S-transferase and chalcone synthase in tobacco and soybean plants and cell cultures. It is concluded that sojeins contribute to the induction of defence responses but that they are not involved in race specific recognition of the P. sojae races by soybean plants. Zusammenfassung Klonierung, Expression und Charactier von Proteinelictoren aus dem Soyabohnenpathogen Phytophthora sojae Der Oomycet Phytophthora sojae ist ein ernstes Pathogen der Sojabohne. In der Sojabohne gibt es mehrere Resistenzgene gegen verschiedene Rassen von P. sojae, jedoch ist die Natur der korrespondierenden Avirulenzgene unbekannt. Wir haben 4 verschiedene Isoformen eines Protein-Elicitors aus P. sojae (Sojein 1,4) kloniert, die zur Klasse der sauren ,-Elicitine gehören. Sie kodieren für Proteine mit 98 Aminosäuren und zeigen hohe Homologie zu Elicitinen aus anderen Phytophthora Spezies. Aus genomischer DNA und aus revers-transkribierter mRNA wurden die gleichen 4 Isoformen erhalten. Die verschiedenen Sojeine wurden in Escherichia coli als His-markierte Fusionproteine exprimiert. Sowohl gereinigtes als auch rekombinantes Sojein induziert HR-ähnliche Läsionen in Tabak. In der Sojabohne sind sie allerdings nicht als rassenspezifische Elicitoren aktiv. Dagegen induzieren alle Sojein-Isoformen Abwehrgene wie die Phenylalanin Ammonium-Lyase, Glutathion-S-Transferase und Chalkonsynthase in Tabak-und Sojabohnenpflanzen und Zellkulturen. Die Sojeine tragen also zur Induktion von Abwehrreaktionen bei, sind aber nicht in die rassenspezifische Erkennung von P. sojae durch Sojabohnenpflanzen involviert. [source] Phenols in spikelets and leaves of field-grown oats (Avena sativa) with different inherent resistance to crown rust (Puccinia coronata f. sp. avenae)JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 11 2009Lena H Dimberg Abstract BACKGROUND: Avenanthramides, health-beneficial phenols in oats, are produced in response to incompatible races of the crown rust fungus, Puccinia coronata, in seedlings of greenhouse-grown oats. This study aimed to elucidate whether avenanthramides and/or other phenolic compounds, together with the activities of phenylalanine ammonia lyase (PAL), phenoloxidase (PO) and the avenanthramide biosynthetic enzyme hydroxycinnamoyl-CoA:hydroxyanthranilate- N -hydroxycinnamoyl transferase (HHT), are associated with crown rust infection in mature field-grown oats. Nine oat (Avena sativa L.) genotypes with wide variation in crown rust resistance were exposed to naturally occurring fungal spores during the growth period. RESULTS: In the spikelets avenanthramides as well as HHT activities were more abundant in the crown rust resistant genotypes, whereas p -coumaric and caffeic acids were more abundant in the susceptible ones. In the leaves avenanthramides were not associated with resistance. Instead two unknown compounds correlated negatively with the rust score. Phenols released by alkaline hydrolysis and PAL and PO activities were not related to rust infection, either in spikelets or in the leaves. CONCLUSION: Because grains of crown rust-resistant oat genotypes seemed to have higher endogenous levels of health-promoting avenanthramides, use of resistant oats may contribute to a food raw material with health-beneficial effects. Copyright © 2009 Society of Chemical Industry [source] Soil moisture stress-induced alterations in bioconstituents determining tea qualityJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 12 2003PR Jeyaramraja Abstract The impact of water stress on the biochemical constituents that determine black tea quality was investigated. Phenylalanine ammonia lyase (PAL) activity was highest in the drought tolerant ,Assam' cultivar UPASI-2, followed by UPASI-8 and UPASI-9, under non-stress conditions. Under soil moisture stress a reduction in PAL activity was found in all three clones investigated. A strong positive correlation was observed between an increase in soil moisture deficit and a decrease in PAL activity. Lower PAL activity correlated well with lower synthesis of flavanols such as epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), which are important precursors of theaflavin-3,3,-digallate that determines final tea quality. Altered synthesis of EGCG and ECG could be due to their molecular rearrangement at elevated leaf temperature during drought. Synthesis of quality constituents such as gallic acid and caffeine declined significantly owing to both drought and waterlogging stress. The reduction in gallic acid due to water stress could lead to lower synthesis of theaflavin fractions such as epitheaflavic acid, epitheaflavic acid-3,-gallate and theaflavic acid and, thereby, quality deterioration. Similarly to drought, flooding stress was also found to alter the biochemical constituents necessary for tea quality. Copyright © 2003 Society of Chemical Industry [source] Characteristics of murine histidinaemia and its potential for genetic manipulationLIVER INTERNATIONAL, Issue 4 2004N. Mellor Abstract: Background: Histidinaemia is an autosomal recessive disorder affecting the hepatic enzyme histidine ammonia lyase (histidase) resulting in elevated plasma and urinary histidine and is prototypic of a series of hepatic cytosolic enzyme defects. Aims: To characterise the physiology of murine histidinaemia with respect to histidine excretion and catabolism, and explore the potential for manipulating cellular and whole body histidase metabolism by gene transfer. Materials and Methods: We studied his/his mice which have a G to A substitution in the gene encoding histidase, using both in vitro transduction of isolated hepatocytes by lipofection with wild-type histidase cDNA, and in vivo transduction of whole liver using a retroviral construct. Results and Conclusion: Histidase cDNA expression restored histidase activity in vivo and in vitro towards normal levels, demonstrated both at the cellular level and by whole body metabolic studies, establishing the potential of this model for the development of new gene therapeutic approaches. [source] Co-occurring increases of calcium and organellar reactive oxygen species determine differential activation of antioxidant and defense enzymes in Ulva compressa (Chlorophyta) exposed to copper excessPLANT CELL & ENVIRONMENT, Issue 10 2010ALBERTO GONZALEZ ABSTRACT In order to analyse copper-induced calcium release and (reactive oxygen species) ROS accumulation and their role in antioxidant and defense enzymes activation, the marine alga Ulva compressa was exposed to 10 µM copper for 7 d. The level of calcium, extracellular hydrogen peroxide (eHP), intracellular hydrogen peroxide (iHP) and superoxide anions (SA) as well as the activities of ascorbate peroxidase (AP), glutathione reductase (GR), glutathione-S-transferase (GST), phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX) were determined. Calcium release showed a triphasic pattern with peaks at 2, 3 and 12 h. The second peak was coincident with increases in eHP and iHP and the third peak with the second increase of iHP. A delayed wave of SA occurred after day 3 and was not accompanied by calcium release. The accumulation of iHP and SA was mainly inhibited by organellar electron transport chains inhibitors (OETCI), whereas calcium release was inhibited by ryanodine. AP activation ceased almost completely after the use of OETCI. On the other hand, GR and GST activities were partially inhibited, whereas defense enzymes were not inhibited. In contrast, PAL and LOX were inhibited by ryanodine, whereas AP was not inhibited. Thus, copper stress induces calcium release and organellar ROS accumulation that determine the differential activation of antioxidant and defense enzymes. [source] UV-B-induced DNA damage and expression of defence genes under UV-B stress: tissue-specific molecular marker analysis in leavesPLANT CELL & ENVIRONMENT, Issue 9 2001G. Kalbin Abstract The aim of this study was to investigate the regulatory effect of ultraviolet-B (UV-B) radiation on a number of key stress response genes found in the epidermis and mesophyll of Pisum sativum L., Argenteum mutant. This mutant was chosen for the ease with which the entire epidermis can be removed from the mesophyll tissue. An additional goal was to explore the potential modifying effect of pre-acclimation of plants to UV-B radiation prior to exposure by UV-B during treatment. Results showed that mRNA accumulation was similar during acute short-term UV-B exposure for chalcone synthase (Chs) and short-chain alcohol dehydrogenase (SadA) in both epidermis and mesophyll. In contrast, the mRNA levels differed considerably between tissues for phenylalanine ammonia lyase, chalcone isomerase and lipid transfer protein. After 24 h incubation in visible light after cessation of UV-B exposure, the regulation of mRNA levels also differed between Chs and SadA, the former showing no expression in the epidermis and the latter none in the mesophyll. Acclimation to low UV-B levels before acute exposures resulted in delayed induction of Chs and SadA. Measurements of UV-B-induced cyclobutane pyrimidine dimers (CPDs) showed a greater formation in epidermis than in mesophyll. In addition, acclimation at low UV-B levels resulted in significantly higher basal levels of CPDs than in non-acclimated plants in both mesophyll and epidermis and also in increased damage in concomitant acute exposures. The lack of correlation between the number of CPDs and levels of transcripts for defence genes, indicates that DNA damage does not control transcription of these genes. [source] Alterations in Taxol Production in Plant Cell Culture via Manipulation of the Phenylalanine Ammonia Lyase PathwayBIOTECHNOLOGY PROGRESS, Issue 6 2002Michelle C. Brincat One approach to increasing secondary metabolite production in plant cell culture is to manipulate metabolic pathways to utilize more resources toward production of one desired compound or class of compounds, such as diverting carbon flux from competing secondary pathways. Since phenylalanine provides both the phenylisoserine side chain and the benzoyl moiety at C-2 of Taxol, we speculated that blockage of the phenylpropanoid pathway might divert phenylalanine into Taxol biosynthesis. We used specific enzyme inhibitors to target the first enzyme in the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), the critical control point for conversion of l -phenylalanine to trans -cinnamic acid. Cinnamic acid acted quickly in reducing PAL activity by 40,50%, without affecting total protein levels, but it generally inhibited the taxane pathway, reducing Taxol by 90% of control levels. Of the taxanes produced, 13-acetyl-9-dihydro-baccatin III and 9-dihydrobaccatin III doubled as a percentage of total taxanes in C93AD and CO93P cells treated with 0.20 and 0.25 mM cinnamic acid, when all other taxanes were lowered. The PAL inhibitor ,-aminooxyacetic acid (AOA) almost entirely shut down Taxol production at both 0.5 and 1.5 mM, whereas l -,-aminooxy-,-phenylpropionic acid (AOPP) had the opposite effect, slightly enhancing Taxol production at 1 ,M but having no effect at 10 ,M. The discrepancy in the effectiveness of AOA and AOPP and the lack of effect with addition of phenylalanine or benzoic acid derivatives further indicates that the impact of cinnamic acid on Taxol is related not to its effect on PAL but rather to a specific effect on the taxane pathway. On the basis of these results, a less direct route for inhibiting the phenylpropanoid pathway may be required to avoid unwanted side effects and potentially enhance Taxol production. [source] Engineered Biosynthesis of Phenyl-Substituted PolyketidesCHEMBIOCHEM, Issue 8 2004José Garcia-Bernardo Dr. Polyketide derivatives, such as 1, are obtained in enhanced yield from added benzoate by coexpressing in Saccharopolyspora erythraea a hybrid erythromycin,soraphen polyketide synthase (PKS) together with benzoate:CoA ligase from the enterocin-producing (enc) pathway of a marine streptomycete. Remarkably, coexpression of the single gene encP encoding phenylalanine ammonia lyase is sufficient to produce the PKS primer benzoyl-CoA in S. erythraea from endogenous L -phenylalanine without any need to supply precursors in the medium. [source] Inhibition of Histidine Ammonia Lyase by Heteroaryl-alanines and AcrylatesCHEMISTRY & BIODIVERSITY, Issue 5 2006Adrian Katona Abstract Histidine ammonia lyase (HAL) catalyzes the elimination of ammonia from the substrate to form (E)-urocanate. The interaction between HAL and acrylic acids or alanines substituted with heteroaryl groups in the , -position was investigated. These proved to be strong competitive inhibitors when the heteroaryl groups were furanyl, thiophenyl, benzofuranyl, and benzothiophenyl, carrying the alanyl or acrylic side chains either in 2 or 3 positions, with Ki values between 18 and 139,,M. The exception was (furan-3-yl)alanine which was found to be inert. Tryptophan and 1-methyltryptophan, as well as the corresponding acrylates (=prop-2-enoates), are strong mixed inhibitors of HAL. Theoretically, L -histidine can be dissected into 4-methyl-1H -imidazole and glycine. Whereas these two compounds separately are only very weak inhibitors of HAL, equimolar amounts of both show a Ki value of 1.7±0.09,mM which is to be compared with the Km value of 15.6,mM for the normal reaction. We conclude that 5-methyl-1H -imidazole and glycine mimic the substrate and occupy the active site of HAL in a similar orientation. [source] Two beta-alanyl-CoA:ammonia lyases in Clostridium propionicumFEBS JOURNAL, Issue 3 2005Gloria Herrmann The fermentation of ,-alanine by Clostridium propionicum proceeds via activation to the CoA-thiol ester, followed by deamination to acryloyl-CoA, which is also an intermediate in the fermentation of l -alanine. By shifting the organism from the carbon and energy source ,-alanine to ,-alanine, the enzyme ,-alanyl-CoA:ammonia lyase is induced 300-fold (, 30% of the soluble protein). The low basal lyase activity is encoded by the acl1 gene, whereas the almost identical acl2 gene (six amino acid substitutions) is responsible for the high activity after growth on ,-alanine. The deduced ,-alanyl-CoA:ammonia lyase proteins are related to putative ,-aminobutyryl-CoA ammonia lyases involved in lysine fermentation and found in the genomes of several anaerobic bacteria. ,-Alanyl-CoA:ammonia lyase 2 was purified to homogeneity and characterized as a heteropentamer composed of 16 kDa subunits. The apparent Km value for acryloyl-CoA was measured as 23 ± 4 µm, independent of the concentration of the second substrate ammonia; kcat/Km was calculated as 107 m,1·s,1. The apparent Km for ammonia was much higher, 70 ± 5 mm at 150 µm acryloyl-CoA with a much lower kcat/Km of 4 × 103 m,1·s,1. In the reverse reaction, a Km of 210 ± 30 µM was obtained for ,-alanyl-CoA. The elimination of ammonia was inhibited by 70% at 100 mm ammonium chloride. The content of ,-alanyl-CoA:ammonia lyase in ,-alanine grown cells is about 100 times higher than that required to sustain the growth rate of the organism. It is therefore suggested that the enzyme is needed to bind acryloyl-CoA, in order to keep the toxic free form at a very low level. A formula was derived for the calculation of isomerization equilibra between l -alanine/,-alanine or d -lactate/3-hydroxypropionate. [source] Probing the active site of MIO-dependent aminomutases, key catalysts in the biosynthesis of ,-amino acids incorporated in secondary metabolitesBIOPOLYMERS, Issue 9 2010Heather A. Cooke Abstract The tyrosine aminomutase SgTAM produces (S)-ß-tyrosine from L -tyrosine in the biosynthesis of the enediyne antitumor antibiotic C-1027. This conversion is promoted by the methylideneimidazole-5-one (MIO) prosthetic group. MIO was first identified in the homologous family of ammonia lyases, which deaminate aromatic amino acids to form ,,ß-unsaturated carboxylates. Studies of substrate specificity have been described for lyases but there have been limited reports in altering the substrate specificity of aminomutases. Furthermore, it remains unclear as to what structural properties are responsible for catalyzing the presumed readdition of the amino group into the ,,ß-unsaturated intermediates to form ß-amino acids. Attempts to elucidate specificity and mechanistic determinants of SgTAM have also proved to be difficult as it is recalcitrant to perturbations to the active site via mutagenesis. An X-ray cocrystal structure of the SgTAM mutant of the catalytic base with L -tyrosine verified important substrate binding residues as well as the enzymatic base. Further mutagenesis revealed that removal of these crucial interactions renders the enzyme inactive. Proposed structural determinants for mutase activity probed via mutagenesis, time-point assays and X-ray crystallography revealed a complicated role for these residues in maintaining key quaternary structure properties that aid in catalysis. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 802,810, 2010. [source] | |||||||||||||||||||||||||