Chitosan and chitin oligomers increase phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities in soybean leaves

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and tyrosine ammonia-lyase (TAL, 4.3.1.), the key enzymes of the phenylpropanoid pathway, are inducible in response to biotic (such as chitin from fungal cell walls) and abiotic cues. Application of chitin and chitosan to soybean leaf tissues caused increased activity of PAL and TAL enzymes. The elevation of enzyme activity was dependent on the chain length of the oligomers and time after treatment. The hexamer of chitin and pentamer of chitosan produced the maximum activities at 36 h after treatment as compared to controls. Total phenolic content of soybean leaves increased following chitosan and chitin oligomer treatments, showing a positive correlation between enzyme activity and total phenolic content.     

定向进化改造酪氨酸解氨酶提高大肠杆菌合成对香豆酸产量

对香豆酸是一种具有多种药理活性的天然酚类化合物,也是多种天然药用产物生物合成的前体物质,广泛应用于食品、化妆品、医药等领域。通过微生物合成对香豆酸相对于化学合成和植物提取工艺具有节能减排等优势。但是,目前微生物合成对香豆酸产量较低,难以满足大规模工业发酵生产的要求。为了进一步提高对香豆酸产量,对粘红酵母酪氨酸解氨酶(Tyrosineammonia-lyase,TAL)进行定向进化改造,利用高通量筛选方法从随机突变体文库中筛选TAL催化活性提高的突变体。通过初筛和复筛两轮筛选,从大约10 000个突变体中获得1个TAL催化活性提高1倍的突变体。该突变体包含3个氨基酸突变位点,分别为S9Y、A11N、E518A。进一步通过单点氨基酸饱和突变验证,当S9位点突变为Y、I、N和A11位点突变为N、T、Y时,TAL的催化活性提高1倍以上。通过对S9和A11位点3种类型突变进行组合突变验证,S9Y/A11N和S9N/A11Y突变体的TAL催化活力显著高于其他组合。将S9N/A11Y突变体质粒转入酪氨酸高产菌株CP032。通过摇瓶发酵,该菌株在48 h时的对香豆酸产量达到394.2 mg/L,比对照...     

The effect of herbicides,plant growth regulators and other compounds on phenylalanine ammonia-lyase activity

The in vitro and in vivo effects of a number of herbicides and plant growth regulators on phenylalanine ammonia-lyase (PAL) activity were investigated. The most elective in vitro inhibitors were product analogs, t-cinnamic and p-coumaric acids, and carbonyl reagents, hydroxylamine and nitromethane. Application of the herbicides diuron, dalapon, amiben, and chloropropham, to plants resulted in a decrease in the intracellular concn of PAL. The inhibitory effect of alachlor was found to be dose-responsive and somewhat specific. A correlation between PAL inhibition and herbicidal activity was observed for hydroxylamine. The cytokinin, pyranyl benzyladenine, (PBA) increased PAL activity in pigweed. The possibility of developing herbicides acting through PAL inhibition is discussed.     

The characterization of phenylalanine ammonia-lyase from several plant species

Inhibition of the enzyme phenylalanine ammonia-lyase is considered as a target for the design of herbicides. A reliable and simple assay for the enzyme has been used and the kinetics of the enzyme from several sources compared. Purification of the enzyme from the grass green foxtail (Setaria glauca) did not change its kinetic behavior. The distribution of phenylalanine ammonia-lyase and tyrosine ammonia-lyase activity in various plant species was determined.     

Inhibition of phenylalanine ammonia-lyase by cinnamic acid derivatives and related compounds

Thirty-five derivatives of cinnamic acid and related compounds were tested for inhibition against phenylalanine ammonia-lyase (PAL) derived from sweet potato, pea and yeast. Caffeic and gallic acids showed inhibition against PAL originating from higher plants, but not against yeast PAL. In contrast, yeast PAL was specifically inhibited by p-hydroxycinnamic and p-hydroxybenzoic acids. The results suggest that caffeic and gallic acids may act as regulatory substances in phenylpropanoid metabolism in higher plants. Inhibition experiments with synthetic cinnamic acid derivatives have revealed that the presence of a hydrophobic aromatic ring, α,β-double bond and carboxyl group is essential for inhibitory activity. 2-Naphthoic acid which fulfills these structural requirements showed a strong inhibition. The size and shape of the active site is discussed from structure-activity relationships of cinnamic acid derivatives. o-Chlorocinnamic acid, one of the strongest inhibitors found in this study showed an inhibitory effect on the growth of the roots of rice seedlings.     

Ammonia-lyase and O-methyl transferase activities related to lignification in wheat leaves infected with botrytis

The deposition of lignin around wounds in wheat leaves infected with the non-pathogenic fungus Botrytis cinerea, was preceded by increases in ammonia-lyase and O-methyl transferase activities. The increases were localised in the lignifying tissues. Phenylalanine ammonia-lyase and tyrosine ammonia-lyase increased concurrently and were inseparable by gel filtration or DEAE cellulose chromatography, the two activities probably being the function of a single protein. Infection-specific isoenzymes for either of the ammonia-lyase activities were absent. Increases in caffeic acid O-methyl transferase and 5-hydroxyferulic acid O-methyl transferase activities closely followed those of the ammonia- lyases. It is unlikely that the increased proportion of syringyl groups found in infection-induced lignin is controlled by O-methyl transferase activity. A modified method for the estimation of O-methyl transferase activity in crude wheat extracts is described.     

Further observations on phenylalanine ammonia-lyase in fungi

Phenylalanine ammonia-lyase (PAL) has been detected in an Ascomycete, Nectria cinnabarina. Growth in light increases levels of PAL in some but not all Basidiomycetes.     

Inactive and active alanine 2-oxoglutarate amine transferase in maize embryo

Freshly prepared extracts of maize embryo exhibited a rise of alanine 2-oxoglutarate amine transferase activity when they were treated at temperatures ranging from 35 to 50° before the assay of the enzyme. The same rise of activity was observed when the extracts were incubated at 25° in the presence of alanine. The extracts of maize seeds soaked in the presence of alanine did not show the rise of alanine 2-oxoglutarate amine transferase by heat treatment or incubation with alanine.     

Reappraisal of the inhibitory effect of certain sugars used as osmotica on phenylalanine ammonia-lyase activity

An earlier report that various sugars, especially d-mannitol and d-sorbitol, severely inhibited phenylalanine ammonia-lyase (PAL)¹ activity from tobacco in vitro has been reappraised. This apparent inhibition resulted from complexing of the sugars with borate, used as buffer system in that study, leading to an undetected pH decrease. We find that d-mannitol, d-sorbitol, d-glucose and sucrose are not inhibitory to PAL preparations obtained from sweet-clover leaves or from protoplasts of tobacco and barley, when alternative buffers are used for the assay. Moreover, the inhibition of barley PAL due to borate—sugar complex formation was completely eliminated by readjusting the pH of the reaction mixture to 8.8 before assaying.     

Phytochrome-mediated induction of phenylalanine ammonia-lyase in the cotyledons of tomato (Lycopersicon esculentum Mill.) plants

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.) induction in cotyledons from 96-h dark-grown Lycopersicon esculentum Mill. was studied in response to continuous light and hourly light pulses (blue, red, far red). The increases of PAL promoted by blue and red pulses are reversed completely by immediately following 758 nm irradiations. The response to continuous red light could be substituted for by hourly 6-min red light pulses. The effect of continuous red treatments is mainly due to a multiple induction effect of phytochrome. In contrast to red light, hourly light pulses with far red and blue, light can only partially substitute for continuous irradiation. The continuous blue response could be due to a combination of a multiple induction response and of a high irradiance response of phytochrome. The continuous far red response, could represent a high irradiance response of phytochrome. Dichromatic irradiations indicate that phytochrome is the photoreceptor controlling the light response (PAL) in tomato seedlings.Abbreviations Norflurazon NF-4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-tolyl)-3 (2H) pyridazinone - PAL phenylalanine ammonia-lyase - phytochrome photoequilibrium P_fr/P_tot - P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot total phytochrome: P_r+P_fr     

L-Phenylalanine ammonia-lyase fromPhaseolus vulgaris: Modulation of the levels of active enzyme bytrans-cinnamic acid

The extractable activity ofl-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in cell suspension cultures of bean (Phaseolus vulgaris) is greatly induced following exposure to an elicitor preparation from the cell walls of the phytopathogenic fungusColletotrichum lindemuthianum. Following exogenous application oftrans-cinnamic acid (the product of the PAL reaction) to elicitor-induced cells, the activity of the enzyme rapidly declines. Loss of enzyme activity is accompanied by inhibition of the rate of synthesis of PAL subunits, as determined by [³⁵S]methionine pulse-labelling followed by specific immunoprecipitation; this is insufficient to account for the rapid loss of PAL enzyme activity. Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme. A non-dialysable factor from cinnamicacid-treated bean cells stimulates removal of PAL activity from enzyme extracts in vitro; this effect is dependent on the presence of cinnamic acid. Such loss of enzyme activity in vitro is accompanied by an apparent loss or reduction of the dehydroalanine residue of the enzyme's active site, as detected by active-site-specific tritiation, although levels of immunoprecipitable enzyme subunits do not decrease. Furthermore, cinnamic-acid-mediated loss of enzyme activity in vivo is accompanied, in pulse-chase experiments, by a greater relative loss of³⁵S-labelled enzyme subunits precipitated by an immobilised active-site affinity ligand than of subunits precipitated with anti-immunoglobulin G. It is therefore suggested that a possible mechanism for cinnamic-acid-mediated removal of PAL activity may involve modification of the dehydroalanine residue of the enzyme's active site.Abbreviations AOPP l--aminoxy--phenylpropionic acid - CA trans-cinnamic acid - PAGE polyacrylamide gel electrophoresis - PAL l-phenylalanine ammonia-lyase - SDS sodium dodecyl sulphate     

Behaviour of phenylalanine ammonia-lyase in carrot cells in suspension cultures

The high PAL activity in carrot cells in suspension culture was found at the linear and early stationary phases, with concomitant increases in phenylal     

Biochemical mechanism for the inhibition of phenylalanine ammonia-lyase induction in the absence of oxygen in potato tuber tissue

Induction of phenylalanine ammonia-lyase (PAL) in excised potato parenchyma tissue in the presence of light displayed a rigid requirement for oxygen. A     

Comparison of the properties of histidine ammonia-lyase in normal and histidinemic mutant mice

The histidinemic (his/his) mutant mouse shows greatly reduced skin and liver histidine:ammonia-lyase (HAL; EC 4.3.1.3) activity compared with normal mice. Liver HAL activity in the mutant is heat and salt labile and is inhibited at high substrate concentrations. Two HAL components have been identified in the normal mouse liver, a minor component with properties similar to those of HAL of the mutant mouse and a major component which is heat and salt stable and insensitive to substrate inhibition. Immunotitration with anti-HAL antibody shows that the livers of mutant mice contain no detectable antigenically cross-reacting HAL protein. It is concluded, therefore, that the his allele is a null allele at a structural or regulatory locus for the major HAL enzyme and maps close to the HAL-regulatory locus Hsd and that the low residual HAL activity in the mutant is due to another enzyme.This work was supported by an MRC Project Grant to H.K. and USPHS Grant GH21002 to S.M.A.     

Properties of phenylalanine ammonia-lyase extracted from Cucumis sativus hypocotyls

Some of the in vitro properties of PAL from gherkin hypocotyls were investigated. No metal ion requirement for this enzyme could be demonstrated but there were indications that PAL was a sulphydryl enzyme. Kinetic analysis suggested that PAL exhibited negative homotropic cooperativity. Two K_m values were determined, these were K_m^H, 2·9 × 10^?4 M and K_m^L, 4·3 × 10^?5 M. Strong inhibition of the enzyme was exerted by d-phenylalanine, trans-cinnamic acid, o-coumaric acid, gallic acid, quercetin and kaempferol. Kinetic studies on the inhibition patterns of these compounds established that d-phenylalanine linearized the curvilinear kinetics, trans-cinnamic acid and o-coumaric acid acted as competitive inhibitors whilst gallic acid, quercetin and kaempferol acted as mixed inhibitors. Using a number of desensitization techniques PAL was partially desensitized to inhibition by the mixed inhibitors. These results led to the conclusion that PAL may have a regulatory role in phenol, coumarin and flavonoid biosynthesis.     

A rapid and simplified assay method for tyrosine hydroxylase

Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H2O with the hydroxyl proton of isoamyl alcohol. It is the [3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.     

Control of phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase in gherkin tissues

Blue light mediates a transient increase in the extractable activity of phenylalanine ammonia-lyase from both cotyledons and hypocotyls of etiolated gherkin seedlings, but concurrent changes in extractable cinnamic acid 4-hydroxylase activity only occur in cotyledons. Excision, followed by incubation in the dark, also results in stimulation of the lyase activity in both tissues, but the hydroxylase activity is only stimulated in cotyledons, again concurrently with the lyase. Stimulated levels of hydroxycinnamic acid esters are, however, only formed following light treatment, indicating the presence of another light-sensitive step in their biosynthesis. Treatment of gherkin tissues with 2-aminooxyacetic acid or α-aminooxy-β-phenylpropionic acid inhibits phenylalanine ammonia-lyase activity in situ, reduces the accumulation of hydroxycinnamic acid esters and presumably reduces the endogenous cinnamic acid pool. This treatment increases extractable lyase activity and delays its peak in activity. In cotyledons, these changes in the lyase are associated with concurrent and similar changes in extractable hydroxylase activity, whilst in hypocotyls the hydroxylase is relatively unaffacted. The increase in phenylalanine ammonia-lyase activity following excision of cotyledons and hypocotyls is prevented by cinnamic acid; in cotyledons the hydroxylase is similarly affected, but after a longer lag. Thus whilst cinnamic acid can modify the extractable activity of the lyase, it cannot itself mediate changes in the extractable activity of the hydroxylase.     

苯丙氨酸解氨酶在诱导黄瓜幼苗抗寒性中的作用

为了探讨苯丙氨酸解氨酶(PAL)在诱导黄瓜幼苗抗寒性中的作用,采用喷施特异抑制剂(AOPP)的方法控制PAL活性,测定幼苗抗寒性的变化.结果表明： 低温可以诱导黄瓜幼苗叶片中PAL的基因表达和活性升高;喷施AOPP显著抑制了叶片中PAL活性,减少了酚类和类黄酮物质的积累.低温对黄瓜幼苗造成显著伤害,AOPP预处理加剧了低温对幼苗的损伤,幼苗抗寒性降低.与对照相比,幼苗叶片中相对电解质渗漏率和丙二醛(MDA)含量显著升高,PSII最大光化学效率(F_v/F_m)降低,光化学猝灭参数Y(NO)升高,胁迫相关基因(PR1-1a、COR47、P5CS、HSP70)的诱导表达受到抑制.低温导致黄瓜幼苗叶片中H₂O₂积累,还原型抗坏血酸(AsA)含量降低,脱氢抗坏血酸(DHA)含量升高,AsA∶DHA减小;喷施AOPP的幼苗中抗氧化酶(过氧化氢酶CAT、抗坏血酸过氧化物酶APX)活性显著低于对照,H₂O₂过量积累,AsA∶DHA更低.施用H₂O₂清除剂可以有效缓解喷施AOPP引起的低温损伤加剧,而施用CAT抑制剂的幼苗对低温胁迫更敏感.表明低温诱导了PAL活性升高,促进了苯丙烷类次生代谢产物的合成,提高了胞内抗氧化酶活性,可有效清除活性氧分子,维持AsA氧化还原状态,缓解低温引起的光损伤和氧化损伤.     

Short Report : Effect of amines and guanidines on Rb+ absorption by excised corn roots

The effect of alkyl-amines and -guanidines on the absorption of rubidium by the excised roots of the corn plant was tested. Inhibition of Rb⁺ absorption was observed with both amines and guanidines, where guanidines were more effective. The effect of alkylamines on Rb⁺ transport depends on their molecular structure.