• Abstract and Figures
• Public Full-text

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 31, number 3 FEBS LETTERS May 1973 THREE NOVEL ENZYMES INVOLVED IN THE REDUCTION OF FERULIC ACID TO CONIFERYL ALCOHOL IN HIGHER PLANTS: FERULATE: CoA LIGASE, FERULOYL-CoA REDUCTASE AND CONIFERYL ALCOHOL OXIDOREDUCTASE G.G. GROSS, J’. STGCKIGT, R.L. MANSELL* and M.H. ZENK Lehrstuhl fiir Pjlanzenphysiologie, Ruhr-Universitbt, 463 Bochum, Germany Received 1 March 1973 1. Introduction cambial tissue, of phytotron grown Forsythia sp. were used. The tissue was frozen in liquid nitrogen Using a cell-free system from cambial tissue of and powdered. To the powder was added polyclar AT Sulix alba the reduction of ferulate to coniferyl alco- (w/w) and the mixture subsequently extracted with hol has been shown for the first time, unequivocally, 0.1 M borate buffer pH 7.8 supplemented with lop2 to occur in higher plants [l] . This conversion is de- M 2-mercaptoethanol. After centrifugation the super- pendent on ATP, CoA and reduced pyridine nucleo- natant was fractionated with ammonium sulfate and tides. A reaction sequence has been postulated on the the fraction between 35 and 72% was resuspended in basis of these experiments involving the intermediate the same buffer as above. This solution was treated formation of feruloyl-CoA and coniferyl aldehyde with an anion exchanger and used as a crude enzyme which is in accordance with previous assumptions for source. For the detection of the feruloyl-CoA reduc- this mechanism in lignin biosynthesis as recently re- tase, the above enzyme solution was further fraction- viewed [2]. ated on a Sephadex G-200 column. In the present paper, a cell-free preparation of a Ferulate:CoA ligase was assayed by an optical lignifying higher plant which reduces ferulic acid to method [4] using an c3d5 of 19 X IO6 cm2*mo1e-1. coniferyl alcohol is described. The overall reaction is Spectrophotometric and tracer assays were employed composed of three individual enzymatic steps. Proof for the detection of feruloyl-CoA reductase and conif- is presented that ferulate is first activated by a cinna- eryl alcohol oxidoreductase. mate: CoA ligase to feruloyl-CoA which is in turn re- Coniferyl aldehyde was trapped as an intermediate duced by NADPH to coniferyl aldehyde. This com- of the overall reaction using phenylhydrazine. The al- pound is further reduced to coniferyl alcohol, with dehyde was liberated from the phenylhydrazone by NADPH again participating as the reductant. the addition of excess cu-ketoglutarate, extracted into chloroform, and the 2,4-dinitrophenylhydrazone de- rivative prepared. This derivative was chromato- 2. Materials and methods graphed on thin-layer plates in (I) benzene:ethyl ace- tate, 95:5 and (II) benzene:glacial acetic acid:H20, Radioactive ferulic acid-(0-CH,T) (34 E.cCi/pmole) 6:7:3 (organic phase), and the plates scanned for ra- and coniferyl aldehyde of the same specific activity dioactivity. were prepared [3] . Feruloyl-CoA was prepared and purified as previously described [4] . As an enzyme source young stems, freed of extra- 3. Results * Stipendiat der Alexander von Humboldt Stiftung, on leave 3.1. Ferulate: CoA ligase from Dept. of Biology, University of South Florida, Tampa, Using a crude enzyme preparation from tissue of Fla. 33620, USA. Forsythia our results which had been obtained with North-Holland fiblishing Company - Amsterdam 283 Volume 31, number 3 FEBS LETTERS May 1973 Table 1 Cofactor requirement for ferulate: CoA ligase. Rate relative Feruloyl-CoA to the com- System formed plete system (nmole.min-‘) (%) Complete 1.7 100 minus dithiothreitol 1.5 88 minus Mg’+ 0.7 41 minus ferulate 0 0 minus CoA-SH 0 0 minus ATP 0 0 with heat-denatured enzyme 0 0 I OL m ‘\; 260 300 350 hrn The reaction mixture contained in a final volume of 0.5 ml WAVELENGTH (n m) 25 crmoles Tris-HCl, pH 7.8, 5 pmoles MgClz, 1 pmole dithio- threitol, 0.5 pmoles ferulate, 0.25 pmoles CoA-SH, 1 pmole Fig. 1. UV-spectrum of feruloyl-CoA synthesized under the ATP and 0.17 mg protein. The reaction was followed spectro- catalysis of ferulate:CoA ligase from Forsythia. Solid line: photometrically at 345 nm (temperature 30”). feruloylCoA; dashed line: spectrum after hydrolysis in 0.1 N NaOH. Both spectra recorded in 0.1 M phosphate buffer, pH 7.0. Salix [l] could be confirmed. The Forsythia enzyme had, however, the advantage that the overall reaction The product showed the delayed response with showed an absolute requirement for CoA as well as sodium nitroprusside yielding a yellow color, charac- ATP and reduced pyridine nucleotide in the reduction teristic of the thioester linkage of feruloyl-CoA. Fur- of ferulate to coniferyl alcohol. thermore, the absorption peak at 345 nm disappeared The reduction of ferulate to the corresponding al- upon alkaline hydrolysis (fig. 1) yielding free ferulic cohol is an endergonic process and most likely re- acid as one of the reaction products. Treatment of quires activation of the carboxyl group. The involve- the CoA-ester with 1 M NHzOH at pH 7.0 led also to ment of CoA and ATP strongly suggests the activation the disappearance of the absorption peak at 345 nm. of ferulic acid via a CoA-thiolester. Since the spectra In this case, feruloyl hydroxamate was found to be of CoA-thiolesters of cinnamic acids show a character- the reaction product. On the basis of these experi- istic peak in the near UV at wavelengths between 311 ments we are able to conclude that the intermediate and 363 nm a sensitive optical test can be employed which accumulates under these reaction conditions to detect cinnamate:CoA ligase [4]. This test has and in the absence of reduced pyridine nucleotides, is been used to detect the ligase in our cell-free prepara- feruloyl-CoA. The stoichiometry of the ferulate:CoA tions. As shown in table 1, the activation reaction is ligase reaction will be reported elsewhere. completely dependent on ferulate, CoA and ATP. Mg*+ and a reduced thiol are necessary for full enzy- 3.2. Feruloyl-CoA reductase matic activity. In order to prove the formation of Since feruloyl-CoA has been proven to accumulate, feruloyl-CoA during this reaction, three ml of the in the absence of reduced pyridine nucleotides, the reaction mixture (cf. table 1) were allowed to react possibility existed that either feruloyl-CoA was re- for a period of 120 min. The incubation was worked duced by a specific feruloyl-CoA reductase to conif- up for cinnamoyl-CoA derivatives according to the eryl aldehyde or that it was reduced directly to conif- general method given previously [4] . The purified eryl alcohol. The in vivo findings of Higuchi and product gave the absorption spectrum shown in fig. 1, Brown [.5] would suggest the first possibility, and in with the characteristic absorption maximum at 345 our earlier in vitro experiments [l] , there was evi- nm. This spectrum is identical with that obtained dence that coniferyl aldehyde occurs as an intermedi- from authentic feruloyl-CoA [4]. ate. In order to test this hypothesis, feruloyl-CoA was 284 Volume 31, number 3 FEBS LETTERS May 1973 with authentic material. It is therefore concluded that feruloyl-CoA reductase catalyzes the reduction of feruloyl-CoA to the aldehyde level with NADPH as reductant. Since cinnamoyl-CoA reductase functions at a branch point in phenylpropane metabolism [6] , this enzyme may have important control functions. Studies regarding these regulatory properties are in progress. 4 0 5 10 15 20 3.3. Coniferyl alcohol oxidoreductase TIME (MINI As shown above, a combination of ferulate:CoA Fig. 2. Photometric assay for the reduction of feruloyl-CoA ligase and feruloyl-CoA reductase is involved in the by feruloyl-CoA reductase from Forsythia. The complete reac- formation of coniferyl aldehyde. This compound can tion mixture contained in a final volume of 0.5 ml 50 rmoles Tris-HCl, pH 7.6, 5 pmoles MgCla, 1 rmole dithiothreitol, 20 undergo polymerisation during lignin formation [7] nmoles feruloyl-CoA, 0.2 pmole NADPH and 42 pg protein. or may be reduced to coniferyl alcohol prior to poly- (o-o-o) Complete reaction mixture (NADPH added as in- merisation. This latter step would necessarily involve dicated); (o-o-o) control (feruloyl-CoA omitted, but a cinnamyl aldehyde reductase. NADPH throughout the reaction). Such an enzyme activity was found in the crude extracts. An optical assay based upon the strong ab- incubated in the presence of reduced pyridine nucleo- sorption at 400 nm of coniferyl aldehyde in basic so- tide and crude enzyme. However, under these condi- lution was used. By this method, conifer-y1 alcohol tions, feruloyl-CoA underwent rapid hydrolysis. Thus, oxidoreductase activity could easily be demonstrated the enzyme was further fractionated on a Sephadex in the enzyme extract (table 2). The production of G-200 column and it was possible to remove an appar- coniferyl alcohol from the aldehyde was confirmed as ent thiolesterase activity. A protein fraction was found previously reported [ 1 ] . The enzyme was specific for which catalyzed the reduction of feruloyl-CoA at the NADPH and the reaction was readily reversible. Its expense of NADPH (fig. 2). As indicated, there was substrate specificity is yet unknown, however, it is only minimal thiolesterase activity present and upon most likely that this enzyme is specific for converting addition of NADPH a rapid decrease in optical densi- cinnamyl aldehydes into the immediate lignin precur- ty due to the specific reduction of feruloyl-CoA was sors. It belongs to the large group of aromatic alcohol observed. There was only negligible NADPH oxidase oxidoreductases (cf.

Load more

  4

Copy Link