Purification,cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism

A protein hydrolyzing hydroxycinnamoyl-CoA esters has been purified from tobacco stem extracts by a series of high pressure liquid chromatography steps. The determination of its N-terminal amino acid sequence allowed design of primers permitting the corresponding cDNA to be cloned by PCR. Sequence analysis revealed that the tobacco gene belongs to a plant acyltransferase gene family, the members of which have various functions. The tobacco cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity-purified and cleaved to yield the recombinant enzyme for use in the study of catalytic properties. The enzyme catalyzed the synthesis of shikimate and quinate esters shown recently to be substrates of the cytochrome P450 3-hydroxylase involved in phenylpropanoid biosynthesis. The enzyme has been named hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase. We show that p-coumaroyl-CoA and caffeoyl-CoA are the best acyl group donors and that the acyl group is transferred more efficiently to shikimate than to quinate. The enzyme also catalyzed the reverse reaction, i.e. the formation of caffeoyl-CoA from chlorogenate (5-O-caffeoyl quinate ester). Thus, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase appears to control the biosynthesis and turnover of major plant phenolic compounds such as lignin and chlorogenic acid.     

Distinct substrate specificities and unusual substrate flexibilities of two hydroxycinnamoyltransferases,rosmarinic acid synthase and hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl-transferase,from <Emphasis Type="Italic">Coleus blumei</Emphasis> Benth.

cDNAs and genes encoding a hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase (CbRAS; rosmarinic acid synthase) and a hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (CbHST) were isolated from Coleus blumei Benth. (syn. Solenostemon scutellarioides (L.) Codd; Lamiaceae). The proteins were expressed in E. coli and the substrate specificity of both enzymes was tested. CbRAS accepted several CoA-activated phenylpropenoic acids as donor substrates and d-(hydroxy)phenyllactates as acceptors resulting in ester formation while shikimate and quinate were not accepted. Unexpectedly, amino acids (d-phenylalanine, d-tyrosine, d-DOPA) also yielded products, showing that RAS can putatively catalyze amide formation. CbHST was able to transfer cinnamic, 4-coumaric, caffeic, ferulic as well as sinapic acid from CoA to shikimate but not to quinate or acceptor substrates utilized by CbRAS. In addition, 3-hydroxyanthranilate, 3-hydroxybenzoate and 2,3-dihydroxybenzoate were used as acceptor substrates. The reaction product with 3-aminobenzoate putatively is an amide. For both enzymes, structural requirements for donor and acceptor substrates were deduced. The acceptance of unusual acceptor substrates by CbRAS and CbHST resulted in the formation of novel compounds. The rather relaxed substrate as well as reaction specificity of both hydroxycinnamoyltransferases opens up possibilities for the evolution of novel enzymes forming novel secondary metabolites in plants and for the in vitro formation of new compounds with putatively interesting biological activities.     

Partial Purification and Characterization of Hydroxycinnamoyl-Coenzyme A:Tyramine Hydroxycinnamoyltransferase from Cell Suspension Cultures of Solanum tuberosum

A pathogen elicitor-inducible soluble acyltransferase (tyramine hydroxycinnamoyltransferase [THT], EC 2.3.1), which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-coenzyme A (CoA) esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was partially purified with a 380-fold enrichment and a 6% recovery from cell-suspension cultures of potato (Solanum tuberosum L. cv Datura). The enzyme showed specific activities of 33 mkat (kg protein)-1 (formation of feruloyltyramine). The apparent native Mr was found to be approximately 49,000. Highest activity was at pH 6.8 in K-phosphate. The isoelectric point of the enzyme was approximately pH5.2. The apparent energy of activation was calculated to be 96 kJ mol-1. The enzyme activity was stimulated more than 5-fold by 10 mM Ca2+ or Mg2+. The apparent Km values were 36 [mu]M for feruloyl-CoA and 85 and 140 [mu]M for cinnamoyl- and 4-coumaroyl-CoA, respectively. The Km value for tyramine in the presence of feruloyl-CoA was 22 [mu]M. In the presence of 4-coumaroyl-CoA, however, the Km for tyramine increased to about 230 [mu]M. The mode of action was an iso-ordered bi bi mechanism in which A, B, P, and Q equal hydroxycinnamoyl-CoA, tyramine, N-hydroxycinnamoyltyramine, and CoA, respectively. Thus, the reaction occurred in a ternary complex of the enzyme and substrates. The equilibrium constant of the reaction was determined to be 1.3 x 104. This gave a [delta]G[deg][prime] eq value of -23.5 kJ mol-1.     

Purification of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase from cell-suspension cultures of Solanum tuberosum L. cv. Datura

A pathogen-elicitor-inducible acyltransferase [tyramine hydroxycinnamoyltransferase (THT); EC 2.3.1], which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was purified to apparent homogeneity from cell-suspension cultures of potato (Solanum tuberosum L. cv. Datura), with a 1400-fold enrichment, a 5% recovery and a final specific activity of 208 mkat·(kg protein)^–1. Affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) as eluent was the decisive step in the purification sequence. The purified protein showed a native molecular mass of ca. 49 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and in the absence of a reducing agent (2-mercaptoethanol) indicated that THT is a heterodimer in which the protein subunits (ca. 25 kDa) are non-covalently associated. The enzyme was stimulated fivefold by 10 mM Ca²⁺. The apparent K _m value for tyramine was dependent on the nature of the hydroxycinnamoyl-CoA present. Thus, the K _m value for tyramine was about tenfold greater (174 M) in the presence of 4-coumaroyl-CoA than in the presence of feruloyl-CoA (20 M).Abbreviations PAL phenylalanine ammonia-lyase - THT hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase We thank the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie for financial support. Further support by a grant from the Studienstiftung des Deutschen Volkes to H.H. is gratefully acknowledged.     

Hydroxycinnamoyl-CoA:Putrescine Hydroxycinnamoyltransferase in Tobacco Cell Cultures with High and Low Levels of Caffeoylputrescine

A new hydroxycinnamoyl-CoA:putrescine hydroxycinnamoyltransferase (PHT) was detected in two variant lines of Nicotiana tabacum L. (TX1, TX4) accumulating markedly different levels of caffeoylputrescine. The enzyme accepted only the aliphatic diamines putrescine, cadaverine and 1,3-diaminopropane at a ratio of 100:33:8. Caffeoyl- and feruloyl-CoAs were the best acyl donors. The apparent K_m-values for caffeoyl-CoA and putrescine were near 3 and 10 micromolar, respectively, at the pH-optimum of 10.0. PHT activity was quite similar in low producing TX1 and high producing TX4 cells, while some other biosynthetic enzymes (phenylalanine ammonia-lyase, ornithine decarboxylase) were greatly enhanced in TX4 cells, suggesting that PHT does not catalyze the rate-limiting step in hydroxycinnamoylputrescine formation.     

Induction by light of hydroxycinnamoyl-CoA: Quinate hydroxycinnamoyl transferase in buckwheat (Fagopyrum esculentum Moench): Absence of feed-forward control by trans-cinnamate

The light-induced increase in the activity of hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (CQT) in hypocotyls of etiolated buckwheat seedlings is not suppressed by aminooxy acetic acid, an inhibitor of cinnamic acid formation in this tissue. Incubation of hypocotyls in 2 mmol l^-1 trans-cinnamate in the dark does not increase CQT activity. Thus, trans-cinnamate does not appear to mediate the effect of light on CQT in buckwheat hypocotyls.Abbreviations AOA aminooxy acetic acid - CA4H cinnamic acid-4-hydroxylase - CQT hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase - PAL L-phenylalanine ammonialyase     

Partial purification and properties of hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase from higher plants

The partial purification and characterization of hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (CQT) from two plant sources growing as cell cultures are reported. The enzymes have been purified 50-and 16-fold, respectively, and show an absolute specificity for p-coumaroyl-CoA and caffeoyl-CoA as well as for quinate, and are responsible for the synthesis of p-coumaroylquinate and caffeoylquinate (chlorogenic acid). The distribution of this transferase activity in a variety of plant cell cultures and differentiated plants is reported.     

Enzymic synthesis of caffeoylglucaric Acid from chlorogenic Acid and glucaric Acid by a protein preparation from tomato cotyledons

The phenylpropane metabolism of tomato (Lycopersicon esculentum Mill) cotyledons was investigated. The HPLC analysis revealed two hydroxycinnamic-acid conjugates as major components, identified as chlorogenic acid (5-O-caffeoylquinic acid) and caffeoylglucaric acid (2-O- or 5-O-caffeoyl-glucaric acid). Quantitative analyses indicated a precursor-product relationship between the chlorogenic and caffeoylglucaric acids. Protein preparations from tomato cotyledons were found to catalyze the formation of caffeoylglucaric acid with chlorogenic acid as acyl donor and free glucaric acid as acceptor molecule. This enzyme activity, possibly to be classified as hydroxycinnamoylquinic acid:glucaric acid hydroxycinnamoyltransferase, acts together with hydroxycinnamoyl-CoA: quinic acid hydroxycinnamoyltransferase.     

Release of sphingomyelin phosphodiesterase (acid sphingomyelinase) by ammonium chloride from CL 1D mouse L-cells and human fibroblasts. Partial purification and characterization of the exported enzymes

In cultured human fibroblasts and mouse L-cells the lysosomotropic agent, ammonium chloride, caused release of acid sphingomyelinase into the culture medium. The water-soluble enzymes were partially purified by sequential chromatography on ConA-Sepharose, octyl-Sepharose and Sepharose CL-4B. Mouse sphingomyelinase was purified up to 64-fold and human sphingomyelinase 134-fold from the culture medium. Specific activities were 925 nmol/(h X mg) and 1 434 nmol/(h X mg), respectively. The final enzyme preparations obtained were free of other lysosomal enzyme activities tested and had very similar properties: optimal activity at pH 4.8 (mouse enzyme) and pH 4.4 (human enzyme), Km values of 6.2 X 10(-5)M and 2.4 X 10(-5)M, respectively, and an apparent molecular mass of 68 kDa. In isoelectric focusing the enzymes peaked at pH 4.78 (mouse enzyme) and pH 4.75 (human enzyme).     

Production of caffeic,chlorogenic and rosmarinic acids in plants and suspension cultures of Glechoma hederacea

Glechoma hederacea L. (Lamiaceae) is a perennial plant, which is distributed widely in Europe, Asia and America. Important anti-oxidant compounds are caffeic acid esters like rosmarinic acid (RA) and chlorogenic acid (CA). Phenylalanine ammonia-lyase (PAL) and rosmarinic acid synthase (RAS, 4-coumaroyl-CoA:hydroxyphenyllactic acid hydroxycinnamoyltransferase) contribute to the formation of RA. Our aim in this study was to follow the accumulation of RA, CA and caffeic acid in a suspension culture of G. hederacea. Growth, medium and secondary metabolism parameters were determined during a culture period of 14 days. The maximal PAL activity was observed on day 5 and the maximal RAS activity on day 8. The RA content was exceedingly high and reached 25.9% of the dry mass on day 7. Caffeic acid and CA contents remained rather low. Furthermore, the presence of RA, CA and caffeic acid and the expression patterns of RAS and hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HST), an important enzyme of monolignol formation, in leaves, flowers, stems and roots of naturally grown G. hederacea were assessed. The expression of RAS and HST genes was detectable in all organs except roots. Flowers accumulated 12.5% RA in their dry mass, leaves, stems and roots about 1%. CA was highest in leaves (2.0%), while it was at 1.6% in flowers, 1.3% in stems and almost undetectable in roots. The caffeic acid content remained at or below 0.4% of the dry weight in all organs.     

Elicitor-stimulated biosynthesis of hydroxycinnamoyltyramines in cell suspension cultures of Solanum tuberosum

Treatment of suspension-cultured potato cells (Solanum tuberosum L. cv. Desirée) with an elicitor from Phytophthora infestans induced increased incorporation of 4-hydroxybenzaldehyde, 4-hydroxybenzoate, and N-4-coumaroyl- and N-feruloyltyramine into the cell␣wall and secretion of N-4-coumaroyl- and N-feruloyltyramine into the culture medium. Induced metabolite accumulation was preceded by rapid and transient increases in activities of phenylalanine ammonia-lyase (EC 4.3.1.5) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25), exhibiting maximal activities 5–10 h after initiation of elicitor treatment. Activities of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase (EC 2.3.1.110), catalyzing the formation of N-4-coumaroyl- and N-feruloyltyramine, increased later and remained at high levels. The phenolic defense compounds appear to be involved in cell wall reinforcement and may further directly affect fungal growth in the apoplastic space. Received: 26 July 1997 / Accepted: 9 September 1997     

Effects of competitive inhibitors of phenylalanine ammonia-lyase on the levels of phenylpropanoid enzymes in Solatium tuberosum

Abstract The accumulation of chlorogenic acid in illuminated discs of Solanum tuberosum tuber tissue is accompanied by rapid but transient increases in the activity levels of the biosynthetic enzymes phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase and hydroxycinnamoyl-CoA : quinate hydroxycinna-moyl transferase. Exogenous D-phenylalanine and L-α-aminooxy-β-phenylpropionic acid, competitive inhibitors of phenylalanine ammonia-lyase, inhibit the accumulation of chlorogenic acid and presumably reduce the endogenous pools of pathway intermediates such as cinnamic acid. These treatments prolong the phase of increase in phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase activities and indicate that product feedback modulation is important in maintaining the interrelationship between the levels of these two enzymes during the later stages of induction. In contrast,L-α-aminooxy-β-phenylpropionic acid inhibits the development of hydroxycinnamoyl transferase in illuminated discs supporting the idea that the light-stimulated increase in phenylalanine ammonia-lyase activity causes an increase in cinnamic acid production which mediates the light-stimulated increase in hydroxycinnamoyl transferase activity.     

Induced phenylpropanoid metabolism during suberization and lignification: a comparative analysis

Induction of the biosynthesis of phenylpropanoids was monitored at the enzyme level through measurement of the temporal change in the activity of two marker enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase, (PAL, E.C. 4.1.3.5) and 4-coumaryl-CoA ligase (4-CL, E.C. 6.2.1.12) and two marker enzymes for hydroxycinnamyl alcohol biosynthesis, cinnamoyl-CoA:NADP+ oxidoreductase (CCR, E.C. 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) in both suberizing potato (Solanum tuberosum) tubers and lignifying loblolly pine (Pinus taeda) cell cultures. While measurable activities of PAL, 4-CL and CAD increased upon initiation of suberization in potato tubers, that of CCR did not. By contrast, all four enzymes were induced upon initiation of lignification in pine cell cultures. The lack of CCR induction in potato by wound treatment is consistent with the channelling of hydroxycinnamoyl-CoA derivatives away from monolignol formation and toward other hydroxycinnamoyl derivatives such as those that accumulate during suberization.     

鸭梨PbHCT3基因的克隆及表达分析

羟基肉桂酰CoA:莽草酸/奎宁酸羟基肉桂酰转移酶(HCT)是植物绿原酸合成的关键酶之一.该研究以鸭梨为材料,采用同源基因克隆的方法,克隆出一个鸭梨的HCT基因,命名为PbHCT3.PbHCT3基因cDNA全长序列为1 731bp,基因编码序列长度为1 317bp,编码438个氨基酸,含有酰基转移酶的2个保守序列HHAAD和DFGWG及保守结构域MVVNVTVRES.实时荧光定量PCR分析表明:PbHCT3基因在幼果果皮、果肉、果心、幼叶及花蕾期花瓣中的表达量较高.随着鸭梨果实生长,果实中PbHCT3基因表达量逐渐降低.研究表明,PbHCT3基因与鸭梨幼嫩组织的生长发育有密切关系.     

Dose and Time‐Dependent Effects of Cyanide on Thiosulfate Sulfurtransferase, 3‐Mercaptopyruvate Sulfurtransferase,and Cystathionine λ‐Lyase Activities

We assessed the dose‐dependent effect of potassium cyanide (KCN) on thiosulfate sulfurtransferase (TST), 3‐mercaptopyruvate sulfurtransferase (3‐MPST), and cystathionine λ‐lyase (CST) activities in mice. The time‐dependent effect of 0.5 LD50 KCN on cyanide level and cytochrome c oxidase (CCO), TST, 3‐MPST, and CST activities was also examined. Furthermore, TST, 3‐MPST, and CST activities were measured in stored mice cadavers. Hepatic and renal TST activity increased by 0.5 LD50 KCN but diminished by ≥2.0 LD50. After 0.5 LD50 KCN, the elevated hepatic cyanide level was accompanied by increased TST, 3‐MPST, and CST activities, and CCO inhibition. Elevated renal cyanide level was only accompanied by increased 3‐MPST activity. No appreciable change in enzyme activities was observed in mice cadavers. The study concludes that high doses of cyanide exert saturating effects on its detoxification enzymes, indicating their exogenous use during cyanide poisoning. Also, these enzymes are not reliable markers of cyanide poisoning in autopsied samples.     

Differential effects of light and fungal elicitor on chlorogenic acid and caffeoylshikimic acid metabolism

Upon irradiation an increase in the extractable activity of hydroxycinnamoyl-CoA:d-quinate hydroxycinnamoyl transferase (CQT) and a decrease in the activity of hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (CST) was observed in cell suspension cultures and seedling of carrot (Daucus carota L). Conversely, CST was induced and CQT repressed in the cell cultures upon treatment with fungal elicitor (i.e. cell wall preparations from Phytophthora megasperma). In the cell cultures irradiation led to a continuous accumulation of 5-O-caffeoyl-d-quinic (chlorogenic) acid, while a transient accumulation of 5-O-caffeoylshikimic acid took place in response to elicitor treatment. Cell wall bound 4-hydroxybenzoic, 4-coumaric and also ferulic acid were increased after treatment with Pmg elicitor. These wall bound phenolics may be involved in protection against microbil attack.     

Rosmarinic acid synthase is a new member of the superfamily of BAHD acyltransferases

Purification of rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase) from suspension cells of Coleus blumei Benth. (Lamiaceae) by fractionated ammonium sulphate precipitation, hydrophobic interaction chromatography and two affinity chromatography steps led to the identification of peptide sequences, which enabled a PCR-based approach to isolate the full-length cDNA encoding this enzyme. The open reading frame of the cDNA had a length of 1290 base pairs encoding a protein of 430 amino acid residues with a molecular mass of 47,932 Da with typical characteristics of an acyltransferase of the BAHD superfamily. The cDNA was heterologously expressed in Escherichia coli. The enzyme displayed the activity of rosmarinic acid synthase using 4-coumaroyl- and caffeoyl-coenzyme A and 4-hydroxyphenyllactate as well as 3.4-dihydroxyphenyllactate as substrates. Shikimic acid and quinic acid were not able to serve as hydroxycinnamoyl acceptors. This therefore is the first report of the cDNA-cloning of a rosmarinic acid synthase.