I - PATHWAYS

Chorismate is a key intermediate in the biosynthesis of the shikimic acid pathway, leading to the formation of the aromatic amino acids phenylalanine, tyrosine and tryptophan, as well as a variety of other compounds including the anthraquinones, ubiquinones and folate. These compounds all occur through five distinct biochemical paths, which branch from the point of chorismate, and for this reason, chorismate is often considered a limiting factor in the formation of tryptophan, with much of the compound being utilized for a variety of other metabolites.

PART 1: Chorismate to Indole-3-Glycerol Phosphate

i) Formation of Anthranilate; Characteristics of Anthranilate Synthase:

Scheme 1a: Formation of Anthranilate from Chorismate

As with the rest of the steps involved in the production of tryptophan, this first step in tryptophan synthesis was first characterized in microorganisms, for a variety of reasons that will be explained in the methods section of this paper. However, a variety of sources have noted remarkable similarities in the pathways between the microorganisms and a variety of plants, including Nicotiana tabacum and carrot root (Daucus carota) . In these comparisons, the following basic characteristics of anthranilate synthase occurred in microorganisms, fungi and plants:

- use of glutamine or NH3 as a substrate

- requirement for divalent cations (eg. Mg2+)

- inhibition by the end product of the path, tryptophan

While the actual mechanisms affecting genetic control in enzyme production and organization in organisms have been noted to vary a great deal (the higher plants for example tending to produce more separate genes and multi-functional proteins than microorganisms), the pathway itself seems to be retained throughout. In fact, with more recent studies, sequence determinations of the genes involved in the expression of the enzymes of this path have revealed that a good deal of similarity occurs not only between microorganisms, but even into the fungi and higher plants.

In Escherichia coli (E. coli), Salmonella typhimurium and Aerobacter aerogenes anthranilate synthase occurs as a 2 protein complex with the next enzyme in tryptophan synthesis, anthranilate-5’-phosphoribose-1-pyrophosphate phosphoribosyl transferase (PR transferase), creating a group of polypeptides able to catalyze the first two steps in the path. This was not always the case however, with the enzyme occurring as a single protein unit in Neurospora crassa.

In species where the proteins occurred as a complex, the first component (Anthranilate synthase Component I) was only able to form anthranilate in the presence of high concentrations of ammonia, while the second component (Anthranilate synthase Component II) had full PR transferase activity without the complexion. This indicated that part of the first component’s activity was located in some way in the second, but not vice versa. The two components complexed on their own when mixed in solution.

The inability of component I of anthranilate synthase to catalyze the formation of anthranilate without the second component unless nitrogen was given in the form of NH3 rather than glutamine was explained by H. Zalkin. The second component appeared to be the binding site of glutamine, and it was through this cysteine binding site that the amide nitrogen of glutamine was passed on to the first component as NH3:

Scheme 1b: Detailed Schematic of the Role of Each of the Components of Anthranilate Synthase in Anthranilate Formation

This explained the activity of the first component when fed NH3 as well as its inactivity with the natural substrate without the presence of the second component’s ammonia-producing activity.

In nature, magnesium in its divalent form is required for activity of anthranilate synthase, although it has been shown that other divalent ions, including Co2+ and Mn2+ will also substitute as a cofactor. Some of the heavier divalent cations have an opposite, inhibitory effect on the enzyme however, including Cu2+, Zn2+, Fe2+ and Hg 2+, with Ba2+ having no effect either way.

A feedback mechanism in anthranilate synthase occurs through the final product of the path, tryptophan. The action occurs at the first component of the complex in microorganisms, although it is likely at a site separate from the catalytic site. The activity of this amino acid as an inhibitor was linearly noncompetitive at the same rates whether the substrate used was NH3/NH4+ or glutamine, indicating that glutamine was releasing ammonia to the first component.

The following diagram clearly shows the two separate subunits of anthranilate synthase as a schematic, strands are represented as flattened arrows with the arrowheads at the C terminus and helices are represented as cylinders with points at their C termini:

Figure 1. Schematic Representation of Anthranilate Synthase showing the two separate subunits, Anthranilate Synthase Component I and Anthranilate Synthase Component II.

ii) Formation of N-(5’-Phosphoribosyl) anthranilate; Characteristics of Anthranilate-5’-phosphoribose-1-pyrophosphate phosphoribosyl transferase (PR transferase)

Scheme 2: Formation of N-(5’-Phosphoribosyl)-anthranilate from Anthranilate

The second step in the formation of tryptophan results in the incorporation of the two carbons which will eventually form C-2 and C-3 of the pyrrole ring of indole, from C-1 and C-2 of the phospho-sugar group, 5-phosphoribose-1-pyrophosphate.

The enzyme forming N-(5’-Phosphoribosyl)-anthranilate (PRA) which has already been noted to occur as a protein complex with anthranilate synthase appears to be a less studied enzyme in the tryptophan path, since it occurs mid way through the path and catalyzes a relatively simple reaction. The reaction between ribose-5-phosphate and anthranilic acid actually proceeds through the product of this step, PRA and goes on to rearrange into the third step product, 1-(o-Carboxyphenylamino)-1-deoxyribulose phosphate at room temperature, indicating the extremely low catalytic function of PR transferase and N-(5’-Phosphoribosyl)anthranilate isomerase (PRA isomerase) in this path.

Like anthranilate synthase, PR transferase requires a divalent cation in the form of Mg2+ and it is similarly, although much less inhibited by the final path product, L-tryptophan. One feature tested for was the ability of PR transferase to substitute the high-energy pyrophosphate bond found in the addition substrate, 5-Phosphoribose-1-pyrophosphate with ATP. The author simply replaced this substrate with a combination of ribose-5-phosphate and ATP, which the enzyme was found unable to work with, suggesting that the sugar must be previously phosphorylated by a separate enzymatic pathway before reaching an activated state in regards to PR transferase.

iii)Formation of 1-(o-Carboxyphenylamino)1-deoxyribulose phosphate; Characteristics of N-(5’-Phosphoribosyl)anthranilate isomerase (PRA isomerase)

(iii)A-C - Phosphoribosyl)anthranilate isomerase (PRA isomerase)

Scheme 3: Formation of 1-(o-Carboxyphenylamino)-1-deoxyribulose-5-phosphate from N-(5’-Phosphoribosyl)anthranilate

The nearly irreversible Amadori rearrangement resulting in the formation of 1-(o-Carboxyphenylamino)-1-deoxyribulose-5-phosphate (CdRP) catalyzed by PRA isomerase, while resulting in a single product can be divided into three distinct chemical steps as indicated in Scheme 3. The first step (iii)A results in a ring opening in the sugar, which is followed by a series of two tautomerisms; an imine enamine tautomerism in (iii)B and a keto-enol tautomerism in (iii)C.

Similar to the common occurrence of the protein complex of anthranilate synthase and PR transferase, PRA isomerase is often found in combination with indole-3-glycerol phosphate synthase (InGP synthase). PRA isomerase also occurs in a variety of other forms including a monomeric protein, a single protein with PRA isomerase and InGP synthase activity and as different combinations of multi-catalytic protein complexes with activities outside of the tryptophan biosynthetic pathway.

E. coli is an example of one of the microorganisms with a single protein chain that catalyzes both PRA isomerase and InGP synthase reactions. In this case, the structure appears as two separate domains connected through a polypeptide chain, the PRA isomerase domain occurring at the C-terminal end and the InGP domain occurring at the N-terminal end. The two units, while occurring as well-defined and separate domains, have very similar structural characteristics, both composed of a series of eight-stranded b -barrel strands connected with a -helixes, the catalytic sites both being found in the C-terminal ends of the b -barrels. As with most other bifunctional enzymes, each domain when isolated has much lower activity than when stabilized by their respective domain-domain interactions. The active sites of each of the two domains do not gain any advantage from this proximity, as they face away from each other, PRA isomerase releasing it’s product into the general solvent for InGP synthase to pick up based on concentrations rather than any other mechanism .

iv)Formation of Indole-3-glycerol phosphate; Characteristics of Indole-3-glycerol phosphate synthase

Scheme 4: Formation of Indole-3-glycerol phosphate from 1-(o-Carboxyphenylamino)-1-deoxyribulose-5-phosphate

InGP synthase catalyzes the cyclization of CdRP to C-2 of the original ribose molecule, leading to InGP, with the loss of the carboxyl group of the original anthranilate molecule as carbon dioxide. This reaction which is described as practically irreversible like the Amadori rearrangement in the previous step results in the first product with an indole nucleus.

The enzyme involved in this formation, InGP synthase appears to be as neglected in studies as PRA isomerase, partially due to higher interest by researchers in the remarkably novel features of the first and last enzymes of this path, anthranilate synthase and tryptophan synthase. As well, this reaction is once again a simple one, and has been synthesized in the lab from ribose-5-phosphate and anthranilic acid without any difficulties.

PART 2: Indole-3-Glycerol Phosphate to Tryptophan

v) Formation of Tryptophan; Characteristics of Tryptophan Synthase

Scheme 5: Formation of L-tryptophan from InGP

The formation of L-tryptophan, which stands at the junction between primary and secondary metabolism, via InGP is generally considered in two separate steps based on the separate mechanisms of the intensely scrutinized enzyme, tryptophan synthase. This enzyme has been studied in a wide variety of organisms capable of aromatic amino acid biosynthesis, including the microorganisms (E. coli, Salmonella typhimurium, Synechocystis PCE 6803) and higher plants (Zea mays, Arabidopsis spp., Nicotiana tabacum cell cultures, Pea seedlings and Cicer arictinum).

Early in studies on tryptophan synthase it was discovered that this enzyme was capable of catalyzing three distinct reactions under different conditions :

(1)InGP + L-Ser ŕ L-Trp + glyceraldehyde-3-P

(2)InGP + H2O ß ŕ Indole + glyceraldehyde-3-P

(3)Indole + L-Ser ŕ L-Trp + H2O

Scheme 6: Three Reactions Typical of Tryptophan Synthase

Cancelling H2O and indole out of reactions (2) and (3), results in reaction (1). Since then it has become almost common knowledge with those who study aromatic biosynthesis that tryptophan synthase is composed of a tetramer of two a monomeric subunits in combination with a single b 2 dimeric protein subunit . Due to the relatively large amount of information on tryptophan synthase, each of its subunits will be dealt with individually prior to reconciling their activities together.

The following diagram schematically represents the a and b subunits of a wild-type tryptophan synthase enzyme. The flattened arrows represent strands, with the arrowheads at the C terminal ends and the cylinders represent helices with the points at the C-terminal ends.

Note that this combination of a /b subunits only represents half of the naturally occurring enzyme, with another pair attached through a b 2 protein complex (ie. a b b a ). The a subunit is on the left side of the diagram, the b on the right:

Figure 2: Schematic Representation of Tryptophan Synthase Showing Two of the Subunits.

v)a. Tryptophan Synthase a Subunit:

The a subunit of tryptophan synthase catalyzes the reversible cleavage of InGP to indole and 3-phosphoglyceraldehyde , however this occurs at a much lower rate without complexion to the b 2 subunit, as low as a 1% rate in E. coli. Each a subunit is composed of a single polypeptide chain with no associated metal, which in E. coli measures 267 residues in length and has a mass which typically falls near a MW of 30,000; MW = 28,700 in E. coli and 29,000 in Salmonella typhimurium.

Three dimensional imaging studies on the a subunit of tryptophan synthase revealed that it was smaller than the b 2 subunit and it occurred as an 8-fold a /b barrel, which occurs in a number of other enzyme forms, even occurring in another enzyme of tryptophan biosynthesis, the bifunctional PRA isomerase/InGP synthase.

Substitutions anywhere along the protein chain inactivated the a subunit, and maximum activity was obtained only with two intact cysteine residues. This same study made the possible identification of histidine and methionine residues near the substrate binding site based on correlations between Vmax and pKa values. Later, an arginine residue was identified as the amino acid responsible for the binding of the phosphate of the InGP substrate.

By imaging the enzyme in the presence of a competitive inhibitor structurally similar to InGP, a pit was discovered just below the surface of the subunit filled with hydrophobic residues (Phe-22, Leu-100, Tyr-102, Leu-127, Ala-129, Ile-153, Tyr-175) to bind the indole portion of the natural substrate. This study contradicted the previous discovery of an arginine residue in the binding of the phosphate group, stating that no basic residues were found near the site and instead the phosphate was bound with hydrogen bonding to the backbone of the chain and a serine residue (Ser-235) sidechain. A glutamate group (Glu-49) near the 3’ hydroxyl of bound InGP and an aspartate (Asp-60) whose carboxyl was found to possibly bind with the N atom of the pyrrole ring of InGP both completely inactivated the a subunit of tryptophan synthase when removed, indicating catalytic function.

v)b. Tryptophan Synthase b 2 Subunit:

The b 2 subunit of tryptophan synthase condenses indole with L-serine in the presence of pyridoxal 5’-phosphate (PLP) to form L-tryptophan Each b 2 subunit is a protein dimer with a mass of approximately 100,000 in E. coli. PLP is bound at a ratio of 2 moles of PLP per mole of b 2 subunit dimer and is essential for the activity of both the b 2 subunit in condensing indole with serine and the activity of the entire a 2b 2 complex in forming tryptophan from InGP, but not in the activity of the isolated a subunit.

This cofactor to the b 2 subunit, PLP is used in several other secondary reactions requiring PLP, such as b -eliminations, thiol dependant transaminations and b -c unsaturated amino acid conversions to saturated a -keto acids. This PLP interaction with the b 2 subunit is based on the formation of an azomethine

link with a catalytic lysine residue (Lys-87) which is typical of many decarboxylation reactions:

Scheme 7: The Role of the Catalytic Lysine and PLP in the Formation of an Activated Amino Acid (Ser)

In the case of the b 2 subunit, PLP most likely does not play a role in decarboxylation. The active site contains a lysine residue (Lys-87) which forms a Schiff base with PLP as in Scheme 7, a closely situated histidine (His-82 or His-86) that removes the a -H from the serine to be used in tryptophan formation and an arginine (Arg-148) which bonds to the carboxyl group of the serine. When all of these factors are considered together, it would appear that the transamination occurring between the a amino of the serine and the catalytic lysine residue, eventually leads to Schiff base formation. This probably weakens the bonds to the a -carbon of the serine through electron withdrawal to the PLP positively charged nitrogen, thereby activating it for condensation with the indole molecule in the formation of tryptophan. The hydroxyl group of serine may been replaced with other electronegative groups such as SCH3, OCH3 and Cl without loss of enzymatic action. It would appear that the active site of the b 2 subunit derives its actions by removing the a -hydrogen from serine, which may create an electron deficiency at the methylene group of the serine side chain. This deficiency could explain creation of a new bond between the C-3 atom of the indole substrate and the methylene carbon by an electrophilic attack, with loss of the serine hydroxyl as H2O and formation of tryptophan resulting, although no references to such a mechanism could be found.

Structurally, the b 2 subunit is comprised of two similarly sized domains, both folded into similar helix/sheet/helix structures, the binding site for PLP occurring deep between the two domains. The domains interact over a broad flat surface, with PLP binding deep within this interface.

v)c. Tryptophan synthase a 2b 2 complex:

In its natural form of an a 2b 2 complex, tryptophan catalyzes the overall formation of tryptophan from InGP in the presence of PLP and L-serine, producing D-glycerose-3-phosphate in the process. As with most other protein complexes, the a 2b 2 complex forms readily simply by mixing the two components in aqueous medium and is more stable than either of its isolated subunits. Due to this stability the tetramer catalyzes the overall reaction at a rate increased by 1 to 2 fold in contrast to the individual a and b 2 subunits, however this requires complete physical contact between all the protein components.

When connected, the active site of the a subunit which produces indole is separated from the nearest active b subunit by 25 angstroms, in which case it would seem that the intermediate, indole should appear free in the surrounding substrate. As early as 1958 it was noticed that contrary to the separate reactions catalyzed by each of the subunits, insufficient indole was produced from the a 2b 2 complex to account for the amount of tryptophan produced, this lack of free indole was originally accounted for as an enzyme-substrate complex:

InGP + TSase ŕ Indole-TSase + Triose phosphate

Indole-Tsase + L-Ser ŕ L-Trp + Tsase

Scheme 8: Proposed Mechanism for Lack of Sufficient Free Indole in Tryptophan Formation

For quite some time, the question of why tryptophan synthase did not produce free indole during its production of tryptophan, even when the isolated a subunit did plagued researchers in this field, many researchers repeatedly noting in their work that "…no free intermediates seem likely…" but without any clear evidence as to what the mechanism must be. Eventually, through kinetic studies, three possible explanations were proposed:

    1. A concerted transfer of indole between the active a and b sites.
    2. Covalent attachment throughout the reaction with a concerted mechanism.
    3. Diffusion of free indole between the active a and b sites by a channel of some sort.

Due to the information gathered with three-dimensional structure determinations, the distance between the two active sites was determined as 25 angstroms as previously mentioned. This distance automatically defeated the first two proposed explanations, leaving the third, which was almost immediately confirmed with the images obtained which showed a hydrophobic "tunnel" between the a and b active sites with a diameter large enough to allow passage of indole. Schematically this is represented as follows:

Figure 3: Representation of the hydrophobic tunnels in tryptophan synthase allowing for passage of indole from a sites to b sites without loss of free indole to solvent.

PART 3: Tryptophan to Secondary Metabolic Products

v) Formation of Tryptamine; Characteristics of Tryptophan Decarboxylase

Scheme 9: Formation of Tryptamine from L-Tryptophan

The primary reactions in plants leading to the formation of secondary metabolites from aromatic amino acids usually involve either deamination or decarboxylation. The formation of the indole alkaloids is no exception to this generalization, the first committed step in their formation being the decarboxylation of the primary metabolite, tryptophan to the secondary metabolite, tryptamine by tryptophan decarboxylase. This enzyme has been characterized in cucumber hypocotyls, tomato shoots, barley shoots and Phalaris tuberosa as well as in the terpenoid indole alkaloid (TIA) producing plants, Catharanthus roseus and Camptotheca acuminata. This enzyme, unlike all the other previously mentioned enzymes is not found in all plant species, although it does occur in all indole alkaloid producing species and is one of the possible pathways leading to the formation of the essential plant hormone, indole-3-acetic acid (IAA).

As with all other decarboxylases, tryptophan decarboxylase shows an absolute requirement for PLP and has mechanisms similar to those represented in Scheme 7, the differences being loss of the carboxyl group from the a carbon, rather than the hydrogen and replacement of tryptophan for serine. In Camptotheca acuminata, tryptophan decarboxylase was sequenced and a Pro-His-Lys series of residues was discovered at position 317, this series of residues occurring in all decarboxylases, the lysine residue being the Schiff base forming activating amino acid.

This enzyme is also able to decarboxylate 5-hydroxytryptophan to 5-hydroxytryptamine, but it has no action on D-tryptophan, tyrosine, phenylalanine or 3,4-dihydroxyphenylalanine (DOPA).

Due to its importance as the first committed step in the formation of secondary products based around the indole nucleus, the regulation of tryptophan decarboxylase has been the focus of many studies. This includes its relevance in the production of TIAs, and N,N-dimethyltryptamine (DMT) in Phalaris tuberosa. The studies focussing on tryptophan decarboxylases role in the formation of simple indole alkaloids discovered a fairly large number of compounds which caused inhibition at some level, most of them representing some form of final product:

Table 1: Inhibitors of Tryptophan Synthase

Inhibition of Tryptophan Decarboxylase

Inhibitor

%Inhibition

Competitive Inhibitors:

N,N-dimethyltryptamine

65

Indole-3-acetic acid

60

Inhibitors - Unknown Mechanism

Tryptamine

62

5-Hydroxytryptamine

45

Indole-3-acetaldehyde

50

Non-Inhibitors

5-Methoxy-N,N-dimethyltryptamine

0

5-Methoxytryptamine

0

Indole-3-pyruvic acid

0

Similar studies directed at the TIA producing plants focused on hormonal and genetic controls in an attempt to raise cell cultures with higher alkaloidal contents. Generally, increased auxin concentrations in the medium used to raise the cell cultures resulted in decreased expression of tryptophan decarboxylase transcripts (tdc). Omitting 1-napthaleneacetic acid (NAA) from the medium resulted in a quick rise in tdc mRNA, while addition of extra NAA, IAA or 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in a rapid down-regulation of tdc transcript levels (2,4-D being the only one to cause non-transient reductions).

In many plant species tryptophan decarboxylase expression is strongly regulated by developmental factors, it often occurs in apexes, young stems, young bark and newly germinated shoots. This characteristic will be looked at in more detail in the products section of this paper.

vi) Formation of Strictosidine; Characteristics of Strictosidine Synthase (SSS)

Scheme 10: Formation of Strictosidine from Tryptamine and Secologanin

The formation of strictosidine is the first step in terpenoid indole alkaloid (TIA) production, representing an important combination of terpenoid and indole paths. It occurs through a cyclization starting with formation of a Schiff base between the primary amino group of tryptamine and the formyl group of secologanin. After the formation of this Schiff base, the C-2 of the indole nucleus of tryptamine, which is nucleophilic due to the adjacent nitrogen then attacks the imine carbon in a Pictet-Spengler type reaction to cyclize, followed by loss of the hydrogen at the C-2 position to regain its aromatic status.

All of the monoterpene indole alkaloids are derived from the product of this step, strictosidine, which represents a comparatively large portion of the indole alkaloids, with over 1800 characterized so far, many of them having important medicinal properties, explored in the next section. One of the main reasons that this particular path has been studied is for similar reasons to that of tryptophan decarboxylase; it may represent an important bottleneck in the formation of useful indole alkaloids, such as Corynanthe, Yohimbe, Strychnos, Aspidosperma, Iboga and Hunteria type indole alkaloids. Other examples of combinations of indole moieties with other pathways come from the phenylpropanoids, isoprenoids and polyketides, with a large variety of alkaloids coming from these paths as well. To get an idea of the sheer number and variety of indole alkaloids produced in the natural world, suggested reading is M. Ihara and K Fukumoto’s review, "Recent progress in the chemistry of non-monoterpenoid indole alkaloids" , which includes basic characteristics of the simple alkaloids, non-tryptamines, non-isoprenoid tryptamines, isoprenoid indole alkaloids, ergot alkaloids and bisindole alkaloids.