sign in sign up
<<
Home , Prephenic acid, Shikimic acid

104 4. Biosynthesis of Natural Products Derived from

4.1. Phenyl-Propanoid Natural Products (C6-C3) The biosynthesis of the aromatic amino acids occurs through the shikimic acid pathway, which is found in plants and microorganisms (but not in animals). We (humans) require these amino acids in our diet, since we are unable to produce them. For this reason, molecules that can inhibit on the shikimate pathway are potentially useful as antibiotics or herbicides, since they should not be toxic for humans.

COO

COO NH R = H 3 R = OH R NH3 N H

The aromatic amino acids also serve as starting materials for the biosynthesis of many interesting natural products. Here we will focus on the so-called phenyl-propanoide (C6-C3) natural products, e.g.:

OH OH OH

HO O HO OH HO O

Chalcone OH O a Flavone OH O OH O a Flavonone OH OH Ar RO O O O HO O O OH O OR OH Anthocyanine OH O a Flavonol Podophyllotoxin MeO OMe OMe OH COOH

Cinnamyl alcohol HO O O OH (Zimtsäure) Umbellierfone OH a ) MeO OH O COOH HO Polymerization OH Wood OH HO OH O OH MeO OMe Shikimic acid O HO

4.2. Shikimic acid biosynthesis The shikimic acid pathway starts in carbohydrate . Given the great social and industrial significance of this pathway, the enzymes have been intensively investigated. Here we will focus on the mechanisms of action of several key enzymes in the pathway. The following Scheme shows the pathway to shikimic acid: 105

COO- COO- Phosphoenolpyruvate HO COO- 2- O O3P-O 2- O3P-O DHQ-Synthase 2- O3P-O DAHP-Synthase HO OH O OH O HO OH OH OH 3-Deoxy-D-arabinoheptulo- Dehydroquinate sonate-7-phosphate (DAHP) (DHQ) D-Erythrose-4-phosphate COO- COO- COO- Shikimate Shikimate Dehydroquinase Dehydrogenase

O OH 2- OH HO OH O3P-O OH OH OH Dehydroshikimate Shikimate Shikimate-3-phosphate COO- COO- - 2-O P-O COO- COO OH 3 O COO- EPSP-Synthase Isochorismate 2-O P-O O COO- O COO- 3 Vitamin K OH OH 5-Enolpyruvylshikimate- O Chorismate 3-phosphate (EPSP) HOOC Anthranilate Chorismate COOH Synthase Mutase

COO- COO- ⊕ ⊕ NH OH NH3 3 Aminodeoxychorismate Tryptophan Synthase O COO- Anthranilic acid - COO- COO COO- COO- THF NH HO 3 NH3 O COO- ⊕ ⊕ p-Aminobenzoic acid Tyrosine ⊕ ⊕NH3 NH3 Phenylalanine

DAHP-Synthase At first sight this seems to be a straightforward Aldol-like reaction between phosphoenolpyruvate (PEP) und erythrose-4-phosphate. However, for unknown reasons, Nature has made this more complicated than it appears:

- - O COO O - P DAHP-Synthase COO - O O O 2-O P-O 2-O P-O 3 3 O HO HO OH OH OH

Experiments with 18O-labelled PEP have shown that all of the 18O label is lost with phosphate - none is incorportated into the aldol-product. Other labelling experiments with Z-[3-3H]-PEP have shown that the reaction proceeds stereospecifically, even with respect to the new prochiral center in the product. The Si- face of the PEP must add to the Re-face der carbonyl group. A likely mechanism is : 106

- O - O COO O COO- O O - P -O COO O P - O O O HB - HB 2- O O3P-O HA HA 2- 2-O P-O H O3P-O 3 HO OH O OH HO H HO H OH OH OH

3-Dehydroquinate Synthase This is a very interesting enzymic reaction. At first sight, it is not clear what the reaction mechanism is. The needs NAD+ as coenzyme, but this is not consumed during the reaction (no net redox change):

1 COO- HO COO- 2 O DHQ-Synthase 2- O3P-O 6 4 + 5 NAD HO OH O OH OH OH 3-Deoxy-D-arabinoheptulo- Dehydroquinate (DHQ) sonate-7-phosphate (DAHP)

It was shown that when DAHP is labelled at C5 or C6 with 2H (deuterium), then a significant kinetic isotope effect on the reaction rate can be observed (i.e. slower with the deuterated substrates). This implies that both the C(6)-H and the C(5)-H bonds are cleaved during the reaction. The following mechanism was suggested:

H O HO HO H O HOOC O HOOC O O-P O OH H OH - P O + NADH O H NAD O- HO O OH HO HOOC O HOOC O O H OH

H H HO HO H OH O - DHQ HOOC O HOOC O O OH

This mechanism has been suggested, on the basis of studies carried out over many years. At first sight the enzyme appears to catalyze: 1) a redox reaction, 2) an elimination, 3) another redox reaction, 4) an aldol- like reaction. At least the chemical logic of oxidizing the alcohol group then becomes clear.

How does one active site achieve all this ??

107

Modifications to the phosphate at C-7 have a dramatic effect on rate, suggesting that it plays an active role in the elimination step.

It is known that the labelled substrates 7S- und 7R-[7-3H]-DAHP are converted into labelled products with overall inversion of configuration at C7. So the C-C bond-forming step also proceeds stereospecifically (Proc. Natl. Acad. Sci.USA 1970, 67, 1669). In a model study, however, it was also shown that the the aldol-like reaction can proceed rapidly and also stereospecifically without catalysis by the enzyme (JACS, 1988, 110, 1628):

H H H HO HO o HO H OH hν, 0 C OH OH HOOC O HOOC O HOOC H H O O OH OH D O D D NO2

Apparently, the steps that really need the catalytic action of the enzyme, in order to achieve rapid turnover, are those involving the redox changes (alcohol ketone) with the coenzyme NAD. The catalytic power of the enzyme appears to be focused on making these steps fast, and perhaps is less crucial for providing catalysis for the elimination and aldol-like reactions, which proceed fast anyway if the substrate is bound in an optimal conformation.

EPSP-Synthase The sixth step in shikimic acid biosynthesis is the EPSP-synthase reaction. This enzyme has been intensively investigated, not least because it is the target of the commercially important herbicide , which inhibits the enzyme :

COO- COO- COO- ⊕ 2- + P 2-O P NH O3P-O i 3 2 2- O - O3P-O COO - OH COO 2-O P-O OH 3 5-Enolpyruvylshikimic acid- Glyphosate OH EPSP-Synthase 3-phosphate (EPSP)

Glyphosate is effective in killing a wide variety of plants, including grasses, broadleaf, and woody plants. It has a relatively small effect on some clover species. By volume, it is one of the most widely used herbicides. It is commonly used for agriculture, horticulture, and silviculture, as well as garden maintenance (including home use). Some crops have been genetically engineered to be resistant to glyphosate. Glyphosate was first sold by Monsanto under the tradename "Roundup".

Mechanism of the EPSP synthase reaction ? -- the phosphate group is lost from PEP with cleavage of the C-O bond, not the P-O bond. -- If the enzymic reaction is carried out in D2O, then deuterium is incorporated into the product, and is found equally distributed between the E- and Z-positions in the enolpyruvyl group. 2 2 -- If [3- H2]PEP is used as substrate in H2O then H is lost in equal amounts from the E- und Z- positions in the enolpyruvyl group in the product.

These observations have led to the proposal of an addition-elimination sequence, as shown below:

108

COO COO COO H

CH2 2- O3P-O 2- O 2- OH O3P-O COO O3P-O EPSP-Synthase OH 2 OH OPO3

In one key experiment, the existence of the tetrahedral intermediate was proven. The enzyme (800µM) 13 +S3P (800µM) + 2-[ C]-PEP (1mM) was mixed for 5s, and then quenched with Et3N. exchange chromatography of the resulting products gave a small amount of the intermediate that could be characterized.

Glyphosate is a potent inhibitor of EPSP synthase. The inhibition ist competitive with respect to PEP (Ki = 1µM) but non-competitive with respect to S3P (Eur. J. Biochem. 1984, 143, 351).

E + S ES E + P

E + S ES E + P EI

EI + S ESI

E + S ES E + P

ESI

Crystallographic studies have revealed how the substrate, intermediate, and glyphosate bind at the active site of the enzyme. A substrate analogue Z-3-fluoro-PEP acts as a pseudosubstrate and forms a relatively stable tetrahedral intermediate that could be crystallized on the enzyme (Mol. Microbiol. 2004, 51, 963).

Chorismate Mutase The reaction involves formally a . This reaction occurs at a 1 o measurable rate in aqueous solution even in the absence of the enzyme (t /2 in water at 50 C ≈ 90 min), but the reaction is accelarated about ≈106 fold by the enzyme : 109 COOH COO- Chorismate HOOC Mutase O

O COO- OH Prephenic acid Chorismate OH

The enzymic and the spontaneous reactions could proceed through either chair-like or boat-like transition states. The stereochemical consequences, however, are different: COO

- COO O O COO- COO- boat-like TS

COO- OH OH

O -OOC - HO COO O COO- COO- chair-like TS O COO- OH OH The stereochemical course of both enzymic and spontaneous reactions has been studied, and both have been shown to proceed through chair-like transitions states (JACS, 1984, 106, 2701; JACS 1985, 107, 5306).

Other kinetic and spectroscopic studies have shown that the enzymic reaction most likely is a more-or- less concerted pericyclic reaction. The slowest step appears to be release of product (prephenate) from the enzyme (Biochemistry, 1990, 29, 8872).

Prephenate dehydrogenase and prephenate dehydratase The conversion of prephenate to p-hydroxyphenylpyruvate is catalyzed by the enzyme prephenate dehydrogenase, which requires NAD+. Kinetic isotope studies have suggested that the reaction proceeds in a concerted manner, as shown below :

O OOC COOH Prephenate dehydrogenase COOH O HO HO H N R NAD+ NADH

H2NOC

Finally a transaminase (PLP-dependent) converts the a-ketoacid into the tyrosine. For the production of phenylalanine, the enzyme prephenate dehydratase produces first phenylpyruvate, and then again by transamination the amino acid Phe :

O OOC Prephenate Transaminase COOH COO dehydratase COOH O NH2

HO H

110 Chorismate also plays a key role as precursor to several other very important natural products, including the amino acids tryptophan, p-aminobenzoic acid, as well as p-hydroxybenzoic acid and salicyclic acid.

4.3. , und Lignans

Phenylalanine and tyrosine also act as precursors to a large variety of C6C3-Phenylpropanoide natural products in plants: O

COOH OH OH

NH2 X HO cinnamic acid p-coumaryl alcohol

MeO OH coniferyl alcohol HO Two interesting coumarin derivatives are dicumarol und warfarin, which can prevent blood clotting and are used clinically to treat thrombosis :

Ph O OH HO OH

O O O O O O O O coumarin Warfarin Dicumarol Flavonoids and stilbenes are products from a pathway that uses cinnamoyl-CoA as starter unit, and extends the chain with malonyl-CoA extender units, just like in polyketide biosynthesis. Flavonoids such as Quercitin (in red wine) and catechins (in tea) can act as anti-oxidants. Flavonoids contribute to plant flower colours; yellow from chalcones and flavonols; red, blue and violet from anthocyanidins. Many of these are also found in glycosylated forms in plants. Resveratrol (red wine) has recently been shown to promote longevity in animals:

OH OH OH OH HO O HO O HO O OH OH HO

OH OH OH O OH O OH OH Resveratrol (a stilbene) Naringenin Quercetin (a flavanone) (a flavonol) (an anthocycanidin) Cinnamic acid is also used for the biosynthesis of . Apart from cellulose, lignin is the main component of wood. Lignin is a high molecular weight polymeric material, produced by polymerization of coniferyl alcohol. MeO O O OH OH OH O HO NH 2 MeO O MeO O MeO OMe HO OMe O O OH OH HO O Tyrosine (E)-Coniferyl alcohol MeO OH O O O HO OMe OMe HO OH O O O OMe MeO OMe O OH representative section OH MeO O of a molecule of lignin O MeO O HO Pinoresinol OMe O HO HO O HO

MeO O OMe OH 111 Plant cell walls are complex structures composed mostly of lignocellulose — the most abundant organic material on Earth — a matrix of cross-linked polysaccharide networks, glycosylated proteins, and lignin. This matrix has three main components: cellulose (38–50%), hemicellulose (17–32%), and lignin (15– 30%).

Cellulose is a polysaccharide consisting of a linear chain of several hundred to more than 10,000 D- glucose units linked by β-1,4 bonds. This bonding motif differs from the α-1,4 glucose linkage of starch, such as corn starch that comes from corn kernels.

This structural difference proves to be quite significant. Cellulose chains are linear and somewhat rigid, but starch takes on a coiled chain structure. That makes the cellulose chains amenable to forming numerous hydrogen bonds, which, unlike starch, leads the cellulose chains to assemble into cablelike bundles of crystalline fibrils that have high tensile strength and are resistant to hydrolysis to glucose.

Hemicellulose is also a polysaccharide, but it is typically made up of chains of xylose interspersed with side chains containing arabinose, galactose, mannose, glucose, acetyl, and other sugar groups, depending on plant type. Hemicellulose contains 500 to 3,000 sugar units and includes a small amount of pectin, another polysaccharide, with which it forms a cross-linked network.

Lignin is a cross-linked macromolecule composed of three types of substituted phenols (phenylpropanoids). It fills the spaces in the cell wall between cellulose, hemicellulose, and pectin and is covalently linked to hemicellulose. Lignin resembles a kind of phenol-formaldehyde resin that acts like glue to hold the lignocellulose matrix together. Lignin helps provide additional strength to cell walls and resistance to insects and diseases (C & E News, 2008, Dec. 8, p.15).