Indole-3-glycerol-phosphate synthase

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.

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. 

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

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... 

Indole-3-glycerol-phosphate synthase Confer new catalytic activity (phosphoribosyl anthrani-late isomerase) Rational design + DNA shuffling + selection E. coli [17]... 

Figure 5.41 Early steps of the proposed indole acetic acid biosynthesis pathways for Ara-bidopsis. CHO, chorismate ANA, anthranilate PANA, 5-phosphoribosylanthranilate CADP, l-(o-carboxyphenylamino)-l-deoxyribulose-5-phosphate IGP, indole-3-glycerol phosphate TRP, tryptophan. Enzymes ASA, anthranilate synthetase, suhunit a ASB, anthranilate synthetase, suhunit P PAT, phosphorihosylanthranUate transferase PAI, phosphoiibosylanthrani-late isomerase IGS, indole-3-glycerol-phosphate synthase TSA, tryptophan synthase, subunit a and TSB, tryptophan synthase, suhunit p.

Anthranilate phosphoribosyltransferase 2 phosphoribosyl anthranilate isomerase 3 indole-3-glycerol phosphate synthase 4 tryptophan synthase 25 Luckner, Metabolism... 

Phosphoribosyl anthranilate isomerase Indole glycerol phosphate synthase (E.C. 4.1.2.8)... 

Biosynthesis Like other aromatic amino acids, e.g., Phe and Tyr, Trp is formed on the shikimic acid pathway. There is a branching point at chorismic acid one branch leads to Phe and Tyr, the other to Trp choris-mic acid - anthranilic acid (anthranilic acid synthase, EC 4.1.3.27)- A-(5 -0-phosphoribosyl)-anthranilic acid (anthranilic acid phosphoribosyl transferase, EC 2.4.2.18)- 1 -o-carboxyphenylamino-1 -deoxyribu-lose 5-phosphate [A-(5 -phosphoribosyl)anthranilic acid isomerase]- indole-3-glycerol phosphate (in-dole-3-glycerol phosphate synthase, EC 4.1.1.48) - indole (tryptophan synthase, EC 4.2.1,20)+serine - Trp. Many biologically active indole compounds are derived from Trp, e. g., 5-hydroxytryptophan, 5-hydroxy-tryptamine ( serotonin), and melatonin as well as many indole alkaloids. 

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.

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... 

M. Weyand and I. Schlichting. 1999. Crystal structure of wild-type tryptophan synthase complexed with the natural substrate indole-3-glycerol phosphate S/oc/zemAtry 38 16469-16480. (PubMed)... 

The T, coh enzyme tryptophan synthase, an tetramer, can be dissocia into two a subunits and a P2 dimer (Figure 24.16). 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. Serine forms a Schiff base with this PLP, which is then dehydrated to give the Schiff base of anmioacrylate. This reactive intermediate is attacked by indole to give tryptophan. The overs three-dimensional structure of this enzyme is distinct from that of aspartate aminotransferase and the other PLP enzymes already discussed. 

In B. subtilis, the pathway from chorismate to tryptophan is feedback-inhibited by tryptophan, which suppresses anthranilate synthase activity. Mutant B. subtilis that lacks tryptophan synthetase can grow on minimal medium only when supplemented with exogenous tryptophan. Under these conditions, none of the intermediates in the tryptophan biosynthetic pathway from anthranilate to indole 3-glycerol phosphate are produced. However, when the bacteria have depleted the medium of tryptophan, the levels of those intermediates increase, even though there is no net production of tryptophan. Why ... 

Tryptophan synthase (TS) catalyzes the ultimate step in tryptophan biosynthesis (details see Fig. 4.2). Indole and benzoxazinoid secondary metabolite formation branches from this pathway. The two lyases IGL and BXl cleave indole-3-glycerol phosphate into indole (and glycerolaldehyde-3-phosphate, not shown) and serve as committing enzymes for indole derived secondary metabolites. Indole produced by IGL directly functions as volatile signal. Indole produced by BXl is converted by other enzymes (BX2-BX9) to benzoxazinoids that have an important function in the chemical defense of grasses. 

Figure 4.2 Functional comparison of the indole-3-glycerole phosphate lyases IGL and BXI with the tryptophan synthase complex.

Tryptophan synthase (TS) catalyzes the conversion of indole-3-glycerol phosphate (IGP) and serine to tryptophan. This complex i.s an (ap)2hctcrotctramcr linked via the p-subunits (only one lialf of the TS complex is shown). The cx- and p-subunits catalyze two independent... 

L-Tryptophan synthase, tryptophan desmolase, L-serine hydro-lyase (adding imhleglycerol-phos-phate) (EC 4.2.1.20) the enzyme catalysing the synthesis of L-tryptophan from L-serine and indole 3-glycerol phosphate. T.s. from E. coli (M, 149,000) and other prokaryotes has 02 subunit composition. The enzyme separates easily into monomeric subunit a (also called protein B, M, 29,000) and dimeric subunit 02 (also called protein B Af, of dimer 90,000) when eluted from DEAE cellulose with a sodium chloride gradient. The separated subunits catalyse partial reactions of L-tryptophan synthesis ... 

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