Hans Grisebach

Biosynthesis of Flavonoids

Interest in the biosynthesis of flavonoids was first stimulated by studies on genetic aspects of flower colour (cf. Harborne, 1967; Hess, 1968) and by chemical speculations on the mode of formation of the carbon skeleton of this class of compounds (Birch and Donovan, 1953; Robinson, 1955); tracer studies were first applied to the problem around 1957. Investigations with intact plants or plant tissues led to a basic knowledge of the precursors required and to an understanding of some details of the biosynthesis of flavonoids. From this, a general picture of the interrelationships between various classes of these compounds has emerged. In the course of these tracer experiments, however, it became apparent that a more detailed knowledge of the nature and sequence of the individual biosynthetic steps and their regulation could only be gained by investigations of the enzymes involved.

Photocontrol of flavonoid biosynthesis

The flavonoids are derived from the flavan or isoflavan skeleton (Fig. 1) and comprise a large group of secondary metabolites from higher plants (Harborne et al. 1975). In the literature the term flavonoid is often used to mean all flavonoids except anthocyanins and for the sake of convenience we have followed this usage. Anthocyanins are 3- or 3,5-glycosides of anthocyanidins (Fig. 2) and include the principal red (R), violet and blue (B) plant pigments (λ 520-545 nm).

Regulation of enzyme activities related to the biosynthesis of flavone glycosides in cell suspension culture of parsley (Petroselinum hortense)

Abstract 1. 1. In cell suspension cultures of parsley ( Petroselinum hortense ) the degree of increase of activity of phenylalanine ammonia-lyase caused by white light depends on the growth stage of the cultures. Maximum enzyme stimulation is observed at approximately the tenth day of growth. 2. 2. Changes in the activities of eight enzymes directly related to the biosynthesis of apiin ( 7-O-[β- D -apiofuranosyl -(1 → 2)-β- D -glucosyl ]-5,7,4′- trihydroxyflavone and graveobiosid B (3′-methoxyyapiin) were measured over a period of 24 h after illumination of cell suspension cultures of parsley. 3. 3. The enzymes investigated were phenylalanine ammonia-lyase, trans -cinnamic acid 4-hydroxylase, p -coumarete:CoA ligase, chalcone-flavanone isomerase, UDP-glucose:apigenin 7- O -glucosyltransferase, UDP-apiose:apigenin 7- O -glucoside-[1 → 2]-apiosyltransferase, UDP-apiose synthetasem and S -adenosylmethionine:luteolin3′- O -methyltransferase. 4. 4. Two groups of enzymes could be distinguished on the basis of their responses to light. The first group comprises the three enzymes acting on substrates of the phenylpropanoid type (phenylalanine, cinnamic acid and p -coumaric acid); the second group includes all those enzymes involved exclusively in the formation of flavonoid compounds and their glycosides. 5. 5. A hypothesis is discussed which would correlate three other enzymes assumed to take part in the same biosynthetic pathway with one of these groups of enzymes.

Biosynthesis of cyanidin in cell cultures of Haplopappus gracilis

Abstract -Dihydroquercetin proved to be a good precursor for light-stimulated cyanidin biosynthesis in cell suspension cultures of Haplopappus gracilis . Wit

Substrate specificity of flavanone synthase from cell suspension cultures of parsley and structure of release products in vitro

Abstract The substrate specificity of an extensively purified flavanone synthase from light-induced cell suspension cultures of Petroselinum hortense was investigated. p -Coumaroyl-CoA was found to be the only efficient substrate for flavanone synthesis, producing naringenin (5,7,4′-trihydroxyflavanone). Besides 4-hydroxy-6[4-hydroxystyryl]2-pyrone (F. Kreuzaler and K. Hahlbrock (1975) Arch. Biochem. Biophys. 169 , 84–90) two further release products of the synthase reaction in vitro were identified as 4-hydroxy-5,6-dihydro-6(4-hydroxyphenyl)2-pyrone and p -hydroxybenzalacetone. The apparent K m values for malonyl-CoA and p -coumaroyl-CoA in the reaction leading to naringenin, and for p -coumaroyl-CoA in the reaction leading to the styrylpyrone derivative were 35, 1.6, and 2.6 μ m , respectively. With caffeoyl-CoA as substrate only a very small amount of eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) was formed besides relatively large amounts of the corresponding styrylpyrone, dihydropyrone, and benzalacetone derivatives. No flavanone formation was observed with feruloyl-CoA as substrate, but again appreciable amounts of the three types of short-chain release products were formed. No reaction at all took place with cinnamoyl-CoA, p -methoxycinnamoyl-CoA, isoferuloyl-CoA, or p -hydroxybenzoyl-CoA. None of the styrylpyrone, dihydropyrone, and benzalacetone derivatives has been detected in the cell cultures in vivo . The present results suggest that naringenin is the only natural product of the synthase reaction and that further substitution in the B-ring of the flavonoids occurs in parsley at or after the flavanone stage. The nature of the smaller release products is consistent with the assumption of a stepwise addition of acetate units from malonyl-CoA to the acyl moiety of the starter molecule, p -coumaroyl-CoA.

Coordinated changes in enzyme activities of phenylpropanoid metabolism during the growth of soybean cell suspension cultures

Abstract Variations in teh activities of several enzymes of phenylpropanoid metabolism were studied in fermenter-grown cell suspension cultures of soyben ( Glycine max ). Concomitant large increases and subsequent decreases in the activities of phenylalanine ammonina-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase, and two isoenzymes of p -coumarate:CoA ligase occurred prior to the stationary phase of the cell cultures. These findings represent a further example of an interdependent regulation of these enzymes of the general phenylpropanoid metabolism. The increases in all of these enzyme activities could be further enhanced by illunination of the cells. No comparable light effects and no significant changes were observed for the specific activity of an S -adenosylmethionine: o -dihydric phenol m - O -mehyltransferase and for the overall rate of the two-step reduction of feruloyl-CoA to coniferyl alcohol. These enzymatic reactions therefore appear to be regulated independently of the enzymes of the general phenylpropanoid metabolism.

Induction of phytoalexin synthesis in soybean. Elicitor-induced increase in enzyme activities of flavonoid biosynthesis and incorporation of mevalonate into glyceollin.

Soybean (Glycine max) cotyledons removed from 6-to 8-day-old seedlings were treated with mycelial walls of Phytophthora megasperma var. sojae (elicitor) and the activity changes of the enzymes phenylalanine ammonia-lyase and flavanone synthase were determined. Large increases and subsequent decreases in the activities of both enzymes were found in the elicitor-induced cotyledons. No changes in enzyme activities, occurred in control cut cotyledons with elicitor omitted. Concomitant with the elicitor-stimulated changes in enzyme activities there was accumulation of the soybean phytoalexin, glyceollin. [2-14C] Mevalonic acid was incorporated into glyceollin in elicitor-treated but not in untreated cotyledons. The results prove that stimulation of the enzymes of flavonoid biosynthesis and probably glyceollin biosynthesis are caused by the effect of the P. megasperma elicitor and not merely by wounding the plant material. The induced enzyme levels could control glyceollin biosynthesis.

Enzymic synthesis of lignin precursors: Purification and properties of the S-adenosyl-l-methionine: Caffeic acid 3-O-methyltransferase from soybean cell suspension cultures

Abstract A 3- O -methyltransferase which catalyzes the methylation of caffeic acid to ferulic acid using S -adenosyl- l -methionine as methyl donor has been isolated and purified about 60-fold from cell suspension cultures of soybean ( Glycine max L., var. Mandarin). The enzyme utilized, in addition to caffeic acid ( K m = 133 μM), 5-hydroxyferulic acid ( K m = 55 μM), 3,4,5-trihydroxy-cinnamic acid ( K m = 100 μM), and protocatechualdehyde ( K m = 50 μM) as substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of ferulic acid, of ortho- , meta- , and para -coumaric acids, and of the flavonoid compounds quercetin and luteolin. The methylation of caffeic acid and 5-hydroxyferulic acid showed a pH optimum at 6.5–7.0. No stimulation of the reaction velocity was observed when Mg 2+ ions were added. EDTA did not inhibit the reaction. The K m for S -adencsyl- l -methionine was 15 μ m . S -Adenosyl- l -homocysteine was a potent competitive inhibitor of S -adenosyl- l -methionine ( K i = 6.9 μM).

Biochemie der Flavonoide

AuBerdem erscheinen mineralische Ultrastrukturen im Auflichtbild oft durch organische Substanzen verhtillt. Man wird daher auch weiterhin auf herk6mmliehe Methoden wie Dtinnschliff-Untersuchung und Transmissions-Elektronenmikroskop nicht verzichten, sondern gezielt die jeweils gttnstigsten Methoden einsetzen, um die morphologische Betrachtungsweise auch in der Pal~iontologie fiber den Sichtbereich unseres Auges hinaus zu erweitern. [I] ANGELL, R. W.: J. Protozool. 14, 299 (1967). [2] Bf~, A . H . W . , u. D . B . ERICSOX: Ann. New York Acad. Sci. 109, 65 (t963). [3] H~MLEBEN, CH.: Z i t t e l i ana 1 (1969}. [4] PESSAGNO, E. A., u. K. MIVANO: Micropaleont. 14, 38 (1968). [5] Rz~ss, Z. : l~[icropaleont. 4, 51 (1958). [6] Rzlss , Z.: Bull. Israel geol. Surv. 35, I (1963). [7] S~muT, A . H . : Monogr. brit . Mus. na tur . His t . London, I (1954). [8] TowE, K. IK., u. R. CIFELLI: J. Paleont . 41, 742 (1967).

Enzymatic reduction of (+)-dihydroflavonols to flavan-3,4-cis-diols with flower extracts from Matthiola incana and its role in anthocyanin biosynthesis

A cell-free extract from flowers of Matthiola incana catalyzes a NADPH-dependent stereospecific reduction of (+)-dihydrokaempferol to 3,4-cis-leucopelargonidin (5,7,4′-trihydroxyflavan-3,4-cis-diol). The pH-optimum of this reaction is around 6. The rate of reaction with NADH was about 50% of that found with NADPH. (+)-Dihydroquercetin and (+)-dihydromyricetin were also reduced by the enzyme preparation to the corresponding flavan-3,4-cis-diols. Correlation between the genotype of M. incana and the presence of dihydroflavonol 4-reductase is strong evidence that this enzyme is involved in anthocyanin biosynthesis.