Hispidin biosynthesis in cultures of Polyporus hispidus

Cultures of Polyporus hispidus grown on a liquid medium with glucose as the principal carbon source produced the yellow pigment hispidin (4-hydroxy-6-(3,4-dihydroxystyryl)-2-pyrone) (II). Tracer studies showed that DL-phenylalanine, DL-tyrosine, cinnamate, p-coumarate and caffeate were incorporated into the hispidin molecule without scrambling of the label. Good incorporation of acetate and roalonate into the pyrone portion of the molecule was observed. The related styrylpyrone, bis-noryangonin (4-hydroxy-6-(4-hydroxystyryl)-2-pyrone) (1) was also detected in extracts of cultured mycelium and a cell-free enzyme preparation was obtained which catalyzed the hydroxylation of bis-noryangonin to hispidin.     

O-Methyletransferases endoplasmiques de Populus nigra

O-Methyltransferases catalysing the methylation of caffeic acid to ferulic acid, isoferulic acid and dimethylcaffeic acid were extracted from the endoplasmic reticulum of Populus glandular tissue. The significance of methoxylated cinnamic acids in secreted flavonoid biosynthesis is discussed.     

Metabolism of phaseollin by Colletotrichum lindemuthianum

Two metabolites of the phytoalexin phaseollin (1) produced by cultures of the fungus Colletotrichum lindemuthianum have been assigned structures (3) and (4).     

Metabolism of exogenous adenine by Acer Pseudoplatanus cells

The cells of Acer pseudoplatanus convert erogenous adenine to various metabolites. The balance between synthesis and degradation of adenine nucleotides has been studied for different adenine concentrations and different periods of incubation. The enzymic pathway mediating the synthesis of adenylic nucleotides from erogenous adenine, and those accounting for the degradation of adenine are discussed, and the deamination of AMP as a possible regulatory mechanism governing the size of the pool of adenylic nucleotides is considered.     

转氨酶催化不对称合成芳香族L-氨基酸

芳香族L-氨基酸是合成许多药物、农药、精细化学品和食品添加剂的重要手性砌块(Chiral buildingblocks)。利用酶催化具有高活性和高立体选择性的特点合成手性砌块是目前不对称合成领域重要的研究方向。通过对不同来源转氨酶的进化分析,选择分别源自原核生物大肠杆菌Escherichia coli和真核生物酿酒酵母Saccharomyces cerevisia中的两种具有代表性Ⅰ型芳香族转氨酶TyrB和Aro8,比较研究了两种转氨酶通过平衡逆转不对称氨化催化合成芳香族L-氨基酸的反应过程和催化效率。重组转氨酶TyrB和Aro8都能有效地合成天然芳香族氨基酸苯丙氨酸和酪氨酸以及非天然氨基酸苯甘氨酸。手性HPLC分析表明,合成的氨基酸都是L-构型的,e.e值等于100%。L-丙氨酸是适宜的氨基供体,转氨酶TyrB和Aro8都不能利用D-型氨基酸作为氨基供体。反应体系中氨基供体L-丙氨酸和氨基受体芳香族α-酮酸的最适摩尔比为4∶1。底物芳香族α-酮酸分子结构中芳香环上的取代基以及脂肪酸碳链部分的长度都对酶催化的转氨效率有显著的影响。在制备规模试验中,TyrB催化不对称转氨反应合成L-苯甘氨酸、L-苯丙氨酸和L-酪氨酸的比生产速率为0.28 g/(g.h)、0.31 g/(g.h)和0.60 g/(g.h),Aro8催化上述反应的比生产速率分别为0.61 g/(g.h)、0.48 g/(g.h)和0.59 g/(g.h)。研究结果对利用转氨酶通过平衡逆转不对称催化合成芳香族L-氨基酸的工业化应用具有指导意义。     

The degradation of three-ringed polycyclic aromatic hydrocarbons by wood-inhabiting fungus Pleurotus ostreatus and soil-inhabiting fungus Agaricus bisporus

The degradation of two isomeric three-ringed polycyclic aromatic hydrocarbons by the white rot fungus Pleurotus ostreatus D1 and the litter-decomposing fungus Agaricus bisporus F-8 was studied. Despite some differences, the degradation of phenanthrene and anthracene followed the same scheme, forming quinone metabolites at the first stage. The further fate of these metabolites was determined by the composition of the ligninolytic enzyme complexes of the fungi. The quinone metabolites of phenanthrene and anthracene produced in the presence of only laccase were observed to accumulate, whereas those formed in presence of laccase and versatile peroxidase were metabolized further to form products that were further included in basal metabolism (e.g. phthalic acid). Laccase can catalyze the initial attack on the PAH molecule, which leads to the formation of quinones, and that peroxidase ensures their further oxidation, which eventually leads to PAH mineralization.A. bisporus, which produced only laccase, metabolized phenanthrene and anthracene to give the corresponding quinones as the dominant metabolites. No products of further utilization of these compounds were detected. Thus, the fungi's affiliation with different ecophysiological groups and their cultivation conditions affect the composition and dynamics of production of the ligninolytic enzyme complex and the completeness of PAH utilization.     

Density-labelling studies on the photocontrol of phenylalanine ammonia lyase levels in Cucumis sativus

The de novo synthesis of PAL is demonstrated to occur sometime between imbibition and the end of a 4-hr white light treatment. H₂OD₂O transfer experiments indicate that PAL synthesis may occur during the light period whilst D₂O-H₂O transfer experiments indicate that synthesis of inactive PAL may occur during dark growth followed by activation by light. Neither of these observations is conclusive. De novo synthesis of PAL occurs in excised hypocotyls of gherkin and tuber discs of potato either in darkness or in light. It is concluded that there is as yet no evidence which definitively shows that light controls PAL levels by regulating the rate of de novo synthesis.     

Metabolism of cholesterol by callus culture of Holarrhena antidysenterica

A chemically defined medium was established for the growth of tissue cultures of Holarrhena antidysenterica. Administration of cholesterol-[4-¹⁴C] to 10-day-old callus yielded radioactive 24-methylenecholesterol, 28-isofucosterol, sitosterol, stigmasterol, and conessine, thereby indicating that the conversion of cholesterol into sitosterol is mediated through 24-methylenecholesterol and 28-isofucosterol in this system.     

Biosynthesis of 3,4-dihydroxyphenylalanine in Vicia faba

Radioactive shikimic acid and l-tyrosine were shown to be efficient precursors of 3,4-dihydroxyphenylalanine (DOPA) in Vicia faba. [1-¹⁴C]Acetate and l[U-¹⁴C]phenylalanine were not incorporated into tyrosine or DOPA. Thus the synthesis of DOPA occurs via the shikimic acid pathway and tyrosine or a very closely related metabolise. Phenolase was present in etiolated plants in much larger quantities after a brief light exposure whereas DOPA concentration was relatively constant during all stages of plant growth. Partially purified phenolase did not catalyze the conversion of tyrosine to DOPA and does not appear to have a role in DOPA synthesis.     

Diterpene acids from Helianthus species and their microbiological conversion by Gibberella fujikuroi, mutant B1-41a

The diterpene acid content in 10 species of Helianthus has been investigated. Ent-12,16-cyclokauranoic acid, isolated from H. annuus, is converted into a series of 12,16-cyclogibberellins by cultures of Gibberella fujikuroi, mutant B1-41a, and 12,16-cyclogibberellins A₉, and A₁₂ have been isolated. Ent-12β-acetoxykaurenoic acid and ent-13(S)-angeloxyatisenoic acid have been isolated from H. decapetalus; the metabolism of ent-13(S)-hydroxyatisenoic acid and atisenoic acid by B1-41a is also described.     

苯丙氨酸脱氨酶cDNA在大肠杆菌中的克隆与表达及酶法合成L-苯丙氨酸

利用基因重组技术 ,在大肠杆菌中克隆并表达苯丙氨酸脱氨酶 (PAL) (EC4 .3 .1 .5) ,并应用此酶转化肉桂酸生成L 苯丙氨酸。方法是将欧芹苯丙氨酸脱氨酶cDNA亚克隆到组成型表达载体pMG3 6e启动子P3 2下游 ,以菌落PCR法鉴定插入片段的大小和方向都正确的克隆 ,进而以HPLC检测肉桂酸浓度的方法鉴别重组质粒有催化肉桂酸生成L 苯丙氨酸的酶活力。结果获得能表达PAL酶活性的阳性克隆 ,在pH1 0 ,含 1 .0 %肉桂酸、8.0mol/L氨的转化液中 ,3 0℃反应 2 0h ,肉桂酸重量转化率可达 60 %。该基因工程菌有希望用于工业化生产L 苯丙氨酸。     

Regulation of chorismate mutase activity of various yeast species by aromatic amino acids

The regulatory properties of chorismate mutase, its cellular localization and isoenzyme pattern were investigated in 23 yeast species. All yeasts contained only a single form of the enzyme, which is localized exclusively in the cytosol. The enzyme activity from all sources was activated 3-(Rhodotorula aurantiaca) to 185-fold (Candida maltosa) by tryptophan. The tryphtophan concentration, which was necessary to obtain half maximum velocity was determined to be between 2 (Pichia guilliermondii) and 95 M (Yarrowia lipolytica). Ten yeast species possessed an enzyme that was inhibited by both phenylalanine and tyrosine. The chorismate mutase from four strains was inhibited only by tyrosine and the enzyme from two species was inhibited by phenylalanine alone. The enzyme inhibition by phenylalanine and tyrosine was completely reversed by tryptophan. Six enzyme sources were not inhibited and theY. lipolytica chorismate mutase was slightly activated by both amino acids.     

Quantitative determination of aromatic amino acids and related compounds in rumen fluid by high-performance liquid chromatography

A rapid method for the quantitative determination of tyrosine (Tyr), phenylalanine (Phe), p-hydroxybenzoic acid (HBA), p-hydroxyphenylacetic acid (HPA), benzoic acid (BZA), p-hydroxyphenylpyruvic acid (HPY), phenylacetic acid (PAA), phenyllactic acid (PLA), tryptophan (Trp), indoleacetic acid (IAA), phenylpyruvic acid (PPY), phenylpropionic acid (PPA) and cinnamic acid (CNA) in goat rumen fluid was established by high-performance liquid chromatography (HPLC). The mobile phase used for isocratic elution was 50 mM sodium phosphate buffer (pH 6.5)–methanol (97:3, v/v). The flow-rate was 1.0 ml/min; column temperature 40°C and compounds were monitored at 215 nm with a UV absorbance detector after injection of 10 μl of filtered rumen fluid. Analysis was completed within 40 min. The minimum detectable limits of quantification (μM) of these compounds were Tyr, 2; Phe, 3; HBA, 1; HPA, 2; BZA, 2; HPY, 8; PAA, 3; PLA, 4; Trp, 2; IAA, 2; PPY, 15; PPA, 8 and CNA, 4. Detectable levels of Tyr, Phe, HPA, BZA, HPY, PAA, PLA, Trp and PPA were found in the deproteinized rumen fluid of goat fed a haycube and concentrate mixture. PAA was the predominant compound before and after feeding. The concentrations of HPA, BZA, PAA, PLA and PPA in the goat rumen fluid increased after feeding, while the concentration of Tyr decreased. Phe, HPY and Trp were minor components at all times. PPY, IAA and CNA were not detected and HBA was not completely resolved in the goat rumen fluid.     

Novel metabolic routes during the oxidation of hydroxylated aromatic acids by the yeast Arxula adeninivorans

Aim: To complete our study on tannin degradation via gallic acid by the biotechnologically interesting yeast Arxula adeninivorans as well as to characterize new degradation pathways of hydroxylated aromatic acids. Methods and Results: With glucose‐grown cells of A. adeninivorans, transformation experiments with hydroxylated derivatives of benzoic acid were carried out. The 12 metabolites were analysed and identified by high performance liquid chromatography and GC/MS. The yeast is able to transform the derivatives by oxidative and nonoxidative decarboxylation as well as by methoxylation. The products of nonoxidative decarboxylation of protocatechuate and gallic acid are substrates for further ring fission. Conclusion: Whereas other organisms use only one route of transformation, A. adeninivorans is able to carry out three different pathways (oxidative, nonoxidative decarboxylation and methoxylation) on one hydroxylated aromatic acid. The determination of the K_M‐values for protocatechuate and gallic acid in crude extracts of cells of A. adeninivorans cultivated with protocatechuate and gallic acid, respectively, suggests that the decarboxylation of protocatechuate and gallic acid may be catalysed by the same enzyme. Significance and Impact of the Study: This transformation pathway of protocatechuate and gallic acid via nonoxidative decarboxylation up to ring fission is novel and has not been described so far. This is also the first report of nonoxidative decarboxylation of gallic acid by a eukaryotic micro‐organism.     

Metabolism of aromatic aldehydes as cosubstrates by the acetogen Clostridium formicoaceticum

When the acetogen Clostridium formicoaceticum was cultivated on mixtures of aromatic compounds (e.g., 4-hydroxybenzaldehyde plus vanillate), the oxidation of aromatic aldehyde groups occurred more rapidly than did O-demethylation. Likewise, when fructose and 4-hydroxybenzaldehyde were simultaneously provided as growth substrates, fructose was utilized only after the aromatic aldehyde group was oxidized to the carboxyl level. Aromatic aldehyde oxidoreductase activity was constitutive (activities approximated 0.8 U mg^–1), and when pulses of 4-hydroxybenzaldehyde were added during fructose-dependent growth, the rate at which fructose was utilized decreased until 4-hydroxybenzaldehyde was consumed. Although 4-hydroxybenzaldehyde inhibited the capacity of cells to metabolize fructose, lactate or gluconate were consumed simultaneously with 4-hydroxybenzaldehyde, and lactate or aromatic compounds lacking an aldehyde group were utilized concomitantly with fructose. These results demonstrate that (1) aromatic aldehydes can be utilized as cosubstrates and have negative effects on the homoacetogenic utilization of fructose by C. formicoaceticum, and (2) the consumption of certain substrates by this acetogen is not subject to catabolite repression by fructose. Received: 14 May 1998 / Accepted: 7 August 1998     

Evidence for a pool of inactive phenylalanine ammonia-lyase in Cucumis sativus seedlings

Evidence is presented which suggests that an inactive form of PAL exists in dark grown gherkin seedlings and may be activated by the application of protein synthesis inhibitors. The significance of this finding is discussed with reference to current views on the regulation of PAL activity.     

Ⅱ型抗癌晶体蛋白抗肝癌作用的关键芳香族氨基酸

为探讨Ⅱ型抗癌晶体蛋白(Parasporin-2)上与抗肝癌作用相关的关键氨基酸,利用5-BU对Parasporin-2活性区编码DNA(P2Y)进行PCR诱变,之后在大肠杆菌中表达,产物纯化后经MTT法检测其对肝癌细胞系和正常肝细胞系的作用。获得的9个突变体其抗肝癌活性差异极大,其中P2M1和P2M8对两种肝癌细胞系SMMC7721和Bel7402均有较强的细胞毒杀作用而不影响正常肝细胞系Chang-liver。比较了P2M1、P2M8和P2Y的二级结构与三级结构,发现二级结构上的变化如β折叠变长或α螺旋增加影响着Ⅱ型抗癌晶体蛋白的抗肝癌活性。基于突变体间氨基酸序列比对、突变体与受体间分子对接以及模拟突变等研究的结果表明,位点52、56、58和208上的氨基酸残基特别是芳香族氨基酸在Parasporin-2与受体间的互作中可能起着重要作用。     

Role de la phenylalanine dans la biosynthese des flavonoides et acides “cinnamique” dans les tiges volubiles de Periploca Graeca

Phenylalanine is the precursor of the cinnamic acids and coumarins in the stems and leafs of P. graeca L. Esterification of p-coumaric acid by quinic acid is required before oxidation to chlorogenic acid. In our experiments, we did not obtain radioactive flavonols from ¹⁴C phenylalanine. PAL activity varies as a result of light and temperature in the same manner as the level of flavonoids (especially the phenolic acids). This enzyme, therefore, plays a regulatory role in the synthesis of these phenolic substances. The variation in PAL activity during illumination does not follow the same course as described for other plants.     

Aliphatic and aromatic amines during development of Nicotiana tabacum

Putrescine, spermidine, spermine, tyramine and phenethylamine have been analysed in the apical parts, leaves, stems, flowers and roots of the tobacco p     

Multifaceted plant responses to circumvent Phe hyperaccumulation by downregulation of flux through the shikimate pathway and by vacuolar Phe sequestration

Detrimental effects of hyperaccumulation of the aromatic amino acid phenylalanine (Phe) in animals, known as phenylketonuria, are mitigated by excretion of Phe derivatives; however, how plants endure Phe accumulating conditions in the absence of an excretion system is currently unknown. To achieve Phe hyperaccumulation in a plant system, we simultaneously decreased in petunia flowers expression of all three Phe ammonia lyase (PAL) isoforms that catalyze the non‐oxidative deamination of Phe to trans‐cinnamic acid, the committed step for the major pathway of Phe metabolism. A total decrease in PAL activity by 81–94% led to an 18‐fold expansion of the internal Phe pool. Phe accumulation had multifaceted intercompartmental effects on aromatic amino acid metabolism. It resulted in a decrease in the overall flux through the shikimate pathway, and a redirection of carbon flux toward the shikimate‐derived aromatic amino acids tyrosine and tryptophan. Accumulation of Phe did not lead to an increase in flux toward phenylacetaldehyde, for which Phe is a direct precursor. Metabolic flux analysis revealed this to be due to the presence of a distinct metabolically inactive pool of Phe, likely localized in the vacuole. We have identified a vacuolar cationic amino acid transporter (PhCAT2) that contributes to sequestering excess of Phe in the vacuole. In vitro assays confirmed PhCAT2 can transport Phe, and decreased PhCAT2 expression in PAL‐RNAi transgenic plants resulted in 1.6‐fold increase in phenylacetaldehyde emission. These results demonstrate mechanisms by which plants maintain intercompartmental aromatic amino acid homeostasis, and provide critical insight for future phenylpropanoid metabolic engineering strategies.