Indole and serine

This enzyme [EC 4.1.99.1], also known as L-tryptophan indole-lyase, catalyzes the hydrolysis of L-tryptophan to generate indole, pyruvate, and ammonia. The reaction requires pyridoxal phosphate and potassium ions. The enzyme can also catalyze the synthesis of tryptophan from indole and serine as well as catalyze 2,3-elimination and j8-replacement reactions of some indole-substituted tryptophan analogs of L-cysteine, L-serine, and other 3-substituted amino acids. 

The a subunit catalyzes the formation of indole from indole-3-glycerol phosphate, whereas each P subunit has a PLP-containing active site that catalyzes the condensation of indole and serine to form tryptophan. The overall three-dimensional structure of this enzyme is distinct from that of aspartate aminotransferase and the other PLP enzymes already discussed. Serine forms a Schiff base with this PLP, which is then dehydrated to give the Schiffbase of aminoacrylate. This reactive intermediate is attacked by indole to give tryptophan. 

The conversion of indole to tryptophan has been much more extensively studied. This is brought about by direct reaction of indole and serine under the influence of the enzyme tryptophan desmolase (better named tryptophan synthetase) (302, 853, 854) which requires p5U-idoxal phosphate as a coenzyme (890). The enzyme has been obtained in the cell-free state (890) and partially purified (965) and its genetics in Neurospora studied in detail (966). Zinc appears to play some part in tryptophan desmolase formation or function (628). 

Pyridoxal phosphate is the coenzyme in a large number of amino acid reactions. At this point it is convenient to consider together 1,he mechanism of those pyridoxal-dependent reactions concerned with aromatic amino acids. The reactions concerned are (1) keto acid formation (e.g., from kynurenine, above), 2) decarboxylation (e.g., of 5-hydroxytrypto-phan to 5-hydroxytryptamine, p. 106), (3) scission of the side claain (e.g., 3-tyrosinase, p. 78 tryptophanase, p. 110 and kynureninase, above), and 4) synthesis (e.g., of tryptophan from indole and serine, p. 40). Many workers have considered the mechanism of one or more of these reactions (e.g., 24, 216, 361, 595), but a unified theory is primarily due to Snell and his colleagues (summarized in 593). Snell s experiments have been carried out largely in vitro, and it should be emphasized that in vivo it is the enzyme protein which probably directs the electromeric changes. 

The model system in this study involves the production of tryptophan from indole and serine using tryptophanase (Figure 3). 

Tryptophan synthase (TS) catalyzes the conversion of IGP and serine to tryptophan. The well-characterized bacterial TS enzyme consists of a- and P-subunits that join to form two active sites with a hydrophobic tunnel between them. TS is an a P heterotetramer linked via the P-subunits. The individual subunits catalyze two independent reactions IGP is converted by the a-subunit to indole and glyceraldehyd-3-phosphate, and indole and serine are converted by the p-subunit to tryptophan and H2O. It has been shown for bacterial enzymes that the activity of the isolated subunits is very low in comparison to their activity in the intact TS complex (Table 4.1). Indole is not released from the TS complex but rather travels through the tunnel connecting the active sites of a and P (Fig. 4.2). There is evidence that plant TS, like the bacterial complex, functions as a P heteromers. " The a and P subunits are encoded by independent genes (7X4 and TSB) and the interaction of a and P was inferred from complementation experiments. 

Synthesis of tryptophan from indole and serine has been demonstrated in Neurospora " and E. coli. Further investigation has shown that... 

Evidence for the reaction between indole and serine is that while indole disappears from the culture solutions rather slowly, the addition of serine, and serine only, causes the indole to disappear much more rapidly. Tryptophan accumulates as indole disappears. 

The enzyme which catalyzes the formation of tryptophan from indole and serine has been named tryptophan desmolase. A cell-free preparation has been obtained from Neurospora mycelium which catalyzes this reaction. Pyridoxal phosphate has been found to be a necessary cofactor. The enzyme has been partially purified by Yanofsky. He found the optimum activity to be at pH 7.8, and confirmed the necessity of pyridoxal phosphate as a coenzyme. Effective inhibitors are Co++, Zn++, CN, hydroxylamine, and tryptophan. 

Synthesis of Tryptophan from Indole and Serine, Tatum and Bonner reported that a mutant of Neurospora crassa could utilize indole in place of tryptophan. Umbreit, Wood, and Gunsalus, by using extracts from this mutant, showed that, with pyridoxal phosphate as a coenzyme, tryptophan was synthesized from serine and indole via the following reaction ... 

The reverse reaction, i.e., the breakdown of tryptophan to indole and serine, was not demonstrated with this extract. 

L-Tryptophan is also produced through enzymatic synthesis from indole and serine with the help of tryptophan synthase ... 

Tryptophan synthase catalyzes the last two steps of the biosynthesis of tryptophan. It is typically found as a tetramer. The a subunits catalyze the reversible formation of indole and G3P from InGP. The p subunits catalyze the irreversible condensation of indole and serine to form tryptophan in a PLP-dependent reaction. Each a active site is connected to a p active site in a process known as substrate channeling [18]. Eigure 14.7 represents the stereo view of one ap heterodimer of tryptophan [19]. The a subunit contains bound indole-3-propanol phosphate and p subunit contains L-serine, which forms aldimine with the coenzyme PEP The orange sphere represents the course of tunnel running from the a to the p subunits. This tunnel is a 25 A long hydrophobic channel contained within the enzyme allowing for the diffusion of indole. If the channel did not exist, the indole formed at a active site would quickly diffuse always and be lost to the cell as it is hydrophobic and can easily cross membranes. 

Enzymatic Process. Chemically synthesized substrates can be converted to the corresponding amino acids by the catalytic action of an enzyme or the microbial cells as an enzyme source, t - Alanine production from L-aspartic acid, L-aspartic acid production from fumaric acid, L-cysteine production from DL-2-aminothiazoline-4-catboxyhc acid, D-phenylglycine (and D-/> -hydtoxyphenylglycine) production from DL-phenyUiydantoin (and DL-/)-hydroxyphenylhydantoin), and L-tryptophan production from indole and DL-serine have been in operation as commercial processes. Some of the other processes shown in Table 10 are at a technical level high enough to be useful for commercial production (24). Representative chemical reactions used ia the enzymatic process are shown ia Figure 6. 

This pyridoxal-phosphate-dependent enzyme [EC 4.2.1.20] catalyzes the reaction of L-serine with l-(indol-3-yl)glycerol 3-phosphate to produce L-tryptophan and glyceraldehyde 3-phosphate. The enzyme will also catalyze (a) the conversion of serine and indole into tryptophan and water and (b) conversion of indoleglycerol phosphate into indole and glyceraldehyde phosphate. 

MECHANISM FIGURE 22-18 Tryptophan synthase reaction. This enzyme catalyzes a multistep reaction with several types of chemical rearrangements. An aldol cleavage produces indole and glyceraldehyde 3-phosphate this reaction does not require PLP. Dehydration of serine forms a PLP-aminoacrylate intermediate. In steps and this condenses with indole, and the product is hydrolyzed to release tryptophan. These PLP-facilitated transformations occur at the /3 carbon (C-3) of the amino acid, as opposed to the a-carbon reactions described in Figure 18-6. The /3 carbon of serine is attached to the indole ring system. Tryptophan Synthase Mechanism... 

A (3 replacement reaction catalyzed by the PLP-dependent tryptophan synthase converts indoleglycerol phosphate and serine to tryptophan. Tryptophan synthase from E. coli consists of two subunits associated as an a2P2 tetramer (Fig. 25-3). The a subunit catalyzes the cleavage (essentially a reverse aldol) of indoleglycerol phosphate to glyceraldehyde 3-phosphate and free indole (Fig. 25-2, step s).67 The P subunit contains PLP. It presumably generates, from serine, the Schiff base of aminoacrylate, as indicated in Fig. 25-2 (step f). The enzyme catalyzes the addition of the free indole to the Schiff base to form tryptophan. The indole must diffuse for a distance of 2.5 ran... 

RX— may be a thiol, a hydroxyl, or an indole group. In this way, serine, threonine, cysteine, tryptophan, cystathionine, and serine and threonine phosphates may be interconverted or degraded. 

For several years, indole, which was accumulated by some mutants and used to satisfy the tryptophan requirement by others, was considered an intermediate in tryptophan biosynthesis. Such a role for indole would have been of interest, because it appeared to be an exception to the generalization that biosynthetic intermediates had to bear a charge. It was found that extracts of cells that utilized indole did indeed catalyze the condensation of indole with serine, and extracts of cells that accumulated indole catalyzed the... 

Since tryptophan synthase does not (or poorly) tolerate the substitution of indoles by large groups (iodo- and nitro-groups), such cases have lead to the development of a facial chemical synthesis of racemic A-oc-acetyltryptophan derivatives from 5- and 6-monosubstituted indoles and L-serine, followed by an enzymatic resolution step using acylase Amano resulting in substituted L-tryptophans in high yields and enantiomeric purity of 91-100% ee [66] (Fig. 4). 

This reaction can be considered to be the result of the coupling tryptophanL + h2o = indole + serine and serine = pyruvate + ammonia, and so it involves one constraint. 

Thiostrepton family members are biosynthesized by extensive modification of simple peptides. Thus, from amino acid incorporation studies, the somewhat smaller (mol wt 1200) nosiheptide, which contains five thiazole rings, a trisubstituted indole, and a trisubstituted pyridine, is speculated to arise from a simple dodecapeptide. This work shows that the thiazole moieties arise from the condensation of serine with cysteine (159,160). Only a few reports on the biosynthesis of the thiostrepton family are available (159,160). Thiostrepton is presently used in the United States only as a polyantimicrobial veterinary ointment (Panalog, Squibb), but thiazole antibiotics have, in the past, been used as feed additives in various parts of the world. General (158) and mechanism of action (152) reviews on thiostrepton are available. 

Fig. 11. Channeling in the tryptophan synthase reaction. Indoleglycerol 3-phosphate is cleaved to indole and glyceraldehyde 3-phosphate at the a-site. Indole is then passed through the hydrophobic channel to the /3-subunit where it reacts with serine to form tryptophan. This schematic was drawn from data presented in (55).

Fig. 12. Tryptophan synthase single turnover. A solution of serine and enzyme was mixed with indoleglyerol phosphate (IGP) to initiate the reaction. The disappearance and formation of IGP ( ) and tryptophan (Tip ) were monitored. The reaction was initiated by mixing enzyme (20 /uAf) and serine (10 mM) and radiolabeled IGP (2.4 (jlM). The curves were calculated by computer simulation using the full kinetic pathway (32). For the dashed line, the curves were simulated using a rate of indole binding equal to 20 sec (40 sec > at 20 juM enzyme), predicting a substantial accumulation of indole. Reproduced with permission from (32).

Three features of the reaction kinetics are essential to ensure that indole is channeled efficiently (1) the reaction of serine at the f3 site modulates the formation of indole at the a site such that indole is not produced until serine has reacted to form E AA (2) the rate of reaction of indole and E AA is fast and largely irreversible and (3) the rate of indole diffusion from the a site to the f3 site is very fast (>1000 s ). ° This mechanism accounts for the fact that indole does not accumulate during a single turnover of conversion of IGP into tryptophan (the af3 reaction). This model makes several predictions, which have been tested by kinetic and structural analysis of mutants and alternate substrates ° using single enzyme turnover experiments. 

We now turn to studies with the isolated a protein and ySj protein. The a protein promotes the cleavage reaction of Eqn. 5 at about % of the rate exhibited by the complex, for this conversion. On the other hand the pyridoxal-P containing 2 protein catalyses the condensation reaction between indole and L-serine (Eqn. 6). The efficiency of this process is about 5% of that observed with the intact complex. The 2 protein in the absence of indole also catalyses the deamination reaction ... 

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