Indole glycerol-3-phosphate

Figure 4.6 The bifunctional enzyme PRA-isomerase (PRAI) IGP-synthase (IGPS) catalyzes two sequential reactions in the biosynthesis of tryptophan. In the first reaction (top half), which is catalyzed by the C-terminal PRAI domain of the enzyme, the substrate N-(5 -phosphoribosyl) anthranilate (PRA) is converted to l-(o-carboxyphenylamino)-l-deoxyribulose 5-phosphate (CdRP) by a rearrangement reaction. The succeeding step (bottom half), a ring closure reaction from CdRP to indole-3-glycerol phosphate (IGP), is catalyzed by the N-terminal IGPS domain.

Priestle, J.P, et al. Three-dimensional structure of the bifunctional enzyme N-(5 -phosphoribosyl) anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escheriehia eoli. Proc. Natl. Aead. 

This enzyme [EC 2.4.2.18], also referred to as phospho-ribosyl-anthranilate pyrophosphorylase, catalyzes the reaction of anthranilate with phosphoribosylpyrophos-phate to produce A-5 -phosphoribosylanthranilate and pyrophosphate. In certain species, this enzyme is part of a multifunctional protein, together with one or more other components of the system for the biosynthesis of tryptophan (i.e., indole-3-glycerol-phosphate synthase, anthranilate synthase, tryptophan synthase, and phos-phoribosylanthranilate isomerase). 

Figure 19.24 A real folding funnel. Refolding chromatography of IGPS (49-252) (indole 3-glycerol phosphate synthase lacking residues 1 -48). Denatured IGPS from an inclusion body was dissolved in 8-M urea and diluted onto a 3-mL column of a GroEL minichaperone (GroEF) that was immobilized on agarose. Ninety-six percent of the protein eluted as fully active material.

Indole 3-glycerol phosphate —indole + glyceraldehye 3-phosphate (1,4)... 

Besides having a noncovalent association of subunits as in tryptophan synthase, some enzymes are double-headed, in that they contain two distinct activities in a single polypeptide chain. A good example of this is the indole 3-glycerol phosphate-synthase-phosphoribosyl anthranilate isomerase bifunc-tional enzyme from the tryptophan operon of E. coli. The crystal structure of the complex has been solved at 2.0 A resolution.39 The two enzymes have been separated by genetic manipulation.40 The activity of the two separate monomeric monofunctional constituents is the same as in the covalent complex so there is no catalytic advantage of having the proteins fused. 

The two intermediates in the conversion of anthranilate to indole-3-glycerol phosphate, phosphoribosylan-thranilate and l -(O-carboxyphenylamino)-1 -deoxyribu-lose-5 phosphate, were originally postulated to account for the involvement of phosphoribosyl pyrophosphate in indole-3-glycerol phosphate formation. Support for the postulate was obtained when the dephosphorylated derivative of the second of these intermediates was found in the culture fluids of certain tryptophan-requiring bacterial mutants. The corresponding derivative of the first intermediate has not been found, probably because of its instability. Indeed, this compound, when formed in extracts, is rapidly broken down to anthranilate and ribose-5-phosphate. 

C indole-3-glycerol phosphate synthase none decarboxylative closure of the indole ring... 

The three-dimensional structure of the tryptophan synthase 02)82 complex from S. typhimurium reveals that the four polypeptide subunits are arranged in an extended a/8/Jo order forming a complex 150 A long.7 A schematic view of a single a/ ft pair based on the crystal structure is shown in the color plate . The 02)82 complex catalyzes the synthesis of L-tryptophan from indole-3-glycerol phosphate and L-serine, termed the a)3 reaction (Fig. 7.1). The a and )3 subunits can be separated and shown to catalyze two distinct reactions, termed the a and / reactions, respectively (Fig. 7.1). The rates of the a and / reactions are greatly increased when catalyzed by the 02)82 complex. Although the o)3 reaction is formally the sum of the a and )3 reactions, indole does not appear as a free intermediate in solution in this reaction.17-21 This result indicates that indole is a... 

Fig. 7.1 Reactions catalyzed at the active sites of the a subunit (a reaction) and of the 0 subunit (0 reaction) and the coupled, physiological reaction (a0 reaction). In the a0 reaction, indole produced by cleavage of indole-3-glycerol phosphate at the a site diffuses through an intramolecular tunnel to the 0 site 25-30 A distant where it undergoes a pyridoxal phosphate-dependent /3-replacement reaction with L-serine to form L-tryptophan. Abbreviations used IGP, indole-3-glyceroI phosphate G-3-P, o-glyceraldehyde 3-phosphate, IND, indole [IND], indole intermediate PLP, pyridoxal phosphate.

Fig. 7.10 Cartoon depicting the a/3 reaction. L-serine is converted to the Schiff base of amino acrylate at the j8-site. Indole, which is produced by cleavage of indole-3-glycerol phosphate at the o-site, diffuses through the tunnel and reacts with the Schiff base of amino acrylate at the /3-site to form L-tryptophan. (Reproduced with permission from Brzovic et a .. J. Biol. Chem., 267, 13028 (1992)).

Schiff base, or quinoidal intermediates do not appreciably affect the rate of the a reaction.90113 (2) L-serine and amino acids such as O-methyl-L-serine that form ES III do stimulate the a reaction.113 (3) The rate of indole-3-glycerol phosphate turnover is roughly correlated with the rate of formation of ES III for each of the amino acids.90-113 (4) The kinetics of the lag in cleavage of indole-3-glycerol phosphate and synthesis of L-tryptophan under single turnover conditions correspond to the rate of ES III formation... 

Abbreviations ADCA, adipyl-cephalosporic acid epPCR, error-prone PCR IGPS, indole-3-glycerol phosphate synthase MO, monooxygenase MS, mutator strain PRAI, phosphoribosylanthranilate iso-merase ProFARI, N -[5-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxylamide ribonucleotide isomerase SDM, site-directed mutagenesis, StEP, staggered extension process... 

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