z
Mechanism of lignification
THE origin of lignin is still in doubt, but evidence rhat it arises u l ~ m a t e l y from carbohydrates does exist. Many theories about the nature of its precursors a n d mechanism of its synthesis in plants have been suggested, but most are either speculative or based on indirect and fragmentary evidence ( 6 ) . T h e problem of lignin biogenesib is fundamentally this-how can an aromatic, highly polymerized compound be formed biochemically from substances pre-existing in wood? Investigations of lignin precursors involve considerable difficulty, but biogenesis of the ester, methyl p-methoxycinnamate formed by the wood-destroying mold, Lentinus lepideus, is simpler. 1 n its metabolic processes associated with wood decay, this fungus, a producer of brown rot, forms certain aromatic esters such as methyl cinnamate, methyl p-methoxycinnamate, and methyl anisate. In earlier experiments, this organism was grown on a medium containing glucose, xylose, or ethyl alcohol as the sole carbon source. After several weeks’ growth, methyl p-methoxycinnamate appeared as a crystalline deposit in the culture medium. Therefore, this ester is not a product of lignin degradation (7). These experiments are important if it is assumed that steps in forming this ester are similar to those for lignin (8). This comparison between biogenesis of this ester and lignin is based o n its structural similarity to p-hydroxycinnamyl alcohol, one of the three fundamental precursors of lignin (3). Therefore, the relationship to lignin biogenesis was investigated further. A number of metabolic products of L. Iefiideus were detected (4)-pyruvic acid, acetoacetic acid, oxaloacetic acid, a-ketoglutaric acid, p-hydroxyphenylpyruvic acid, ribose,. glucose, sedoheptulose, and phosphoshikimic acid. Direct origin of methyl p-methoxycinnamate from glucose is indicated because when grown on ethyl alcohol as substrate, the organism resynthesized glucose, and also because of other experiments pre-
viously reported ( 3 ) . This direct conversion then suggested a comparison with biogenesis of certain aromatic amino acids. According to Davis (Z), synthesis of tyrosine occurs via glucose and shikimic acid. Sedoheptulose, which is important in biogenesis of shikimic acid, appears in metabolic products of L. Zepideus. +-Hydroxyphenylpyruvic acid, considered an intermediate in biosynthesis of tyrosine, was also identified in media of this organism and is considered a precursor of p-hydroxycinnamic acid. Thus, a relationship between formation of methyl p-methoxycinnamate and biosynthesis of tyrosine exists. Lignin precursors, because their structural relationship to this ester, may be synthesized in a similar manner. Shikimic acid is regarded as a direct precursor of aromatic rings in the amino acids, phenylalanine, tyrosine, and paminobenzoic acid (2). The following results shows that without rearrangement of the carbon atoms in its sixmembered ring, shikimic acid can also be considered a precursor of aromatic rings in lignin building stones; also, that this transformation parallels the formation mechanism of aromatic amino acids. Specifically labeled shikimic acid was prepared by fermenting 6-C14-n-glucose by Escherichia coli mutant 83-24. In such shikimic acid, 52% of the activity is in position 6 and 43% in position 2 (9). An aqueous solution of this acid was incorporated into the leaves of a fully developed sugar cane plant. The youngest leaves were cut across about 5 cm. from the tips, and the cut ends were dipped immediately into test tubes containing the radioactive material. After several days of continued metabolism, the leaves were removed; the plant stem was cut, dried, pulverized, and thoroughly extracted with water. Activity measurements showed that the sugar cane plant incorporated the radioactive material into nonwater-extractable components of the stem. Counting
the isolated Klason lignin indicated that most radioactivity was concentrated in the lignin because its specific activity was much greater than that of the entire stem. The extracted, dried plant material was then treated with Schweizer’s reagent to remove cellulose, and then with alkaline nitrobenzene (7). The resulting vanillin was isolated and purified by sublimation and by recrystallization ; distribution of radioactivity among the ring carbon atoms in positions 2, 5, and 6 was then determined by three series of degradations. Degradation of the vanillin and subsequent counting of resulting barium carbonate precipitates showed that carbon in position 6 contained 44y0 of the total radioactivity, that in position 2, 41%; and that in position 5, 0%. Thus, distribution of activity in the aromatic ring of vanillin agrees with the original distribution of carbon-14 in the six-membered ring of the incorporated shikimic acid ( 5 ) . I n a similar experiment, carboxyllabeled p-hydroxyphenylpyruvic acid was also incorporated into a mature sugar cane plant, using the same technique as employed with shikimic acid. From these results (70). it appears that most activity of the introduced acid was incorporated into lignin. Hence, it is concluded that, in the biogenesis of lignin itself, p-hydroxyphenylpyruvic acid is an intermediate on the pathway between shikimic acid, derived from carbohydrates and the lignin building stones.
literature Cited (1) Creighton, R., McCarthy, J., Hibbert, H., J . Am. Chem. Sac. 63, 3049 (1941). (2) Davis, B. D., Advances in Enzymol. 16, 247 (1955). (3) Eberhardt, G., J. Am. Chem. SOG.78, 2832 (1956). (4) Eberhardt, G., Nord, F. F., Arch. Biochem. Biophys. 55, 578 (1955). (5) Eberhardt, G., Schubert, W. J., J.Am. Chem. Soc. 78,2835 (1956). ( 6 ) Nord, F. F., Vitucci, J. C., Advances in Enzymol. 8, 253 (1948). (7) Nord, F. F., Vitucci, J. C., Arch. Biochem. 14, 243 (1947). (8) Zbid., 15, 465 (1947). (9) Srinivasan, P. R., Shiguera, H. T., Sprecher, M., Sprinson, D. B., Davis, B. D., J . Biol. Chem. 220, 482 (1956).
(IO) Schubert, W. G., Acerbo, S. N., Nord, F. F., J. Am. Chem. SOC.79, 251 (1957). WALTER J. SCHUBERT and F. F. NORD Department of Organic Chemistry and Enzymology, Fordham University, New York, N. Y. VOL. 49, NO. 9
SEPTEMBER 1957
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