Biosynthesis of Compounds Containing an Indole Nucleus

Parry, Ronald J.

doi:10.1002/9780470186930.ch1

Cited by 3 publications

(6 citation statements)

References 175 publications

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“…The canonical reaction mechanism ( 9 ) proceeds through the sequential steps of condensation, decarboxylation, and dehydration ( Fig. 1 B ).…”

Section: Discussionmentioning

confidence: 99%

“…The names of the decarboxylated substrates are shown in brackets : aniline, phosphoribosylaniline, and PAdRP. B , reaction mechanism of IGPS ( 9 ). The first step is a condensation, where protonation of the ketone by a general acid (HA) results in a carbocation that gets nucleophilically attacked by C1 of the benzyl ring, resulting in intermediate I1.…”

mentioning

confidence: 99%

“…For instance, there are discrepancies in the literature regarding the identities of the catalytic residues and the rate-determining step of the reaction. In 1972, Parry ( 9 ) proposed a reaction mechanism with three sequential steps of condensation, decarboxylation, and dehydration, involving the intermediates I1 and I2 ( Fig. 1 B ), catalyzed by a general acid and a general base.…”

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confidence: 99%

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Structure and kinetics of indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa: Decarboxylation is not essential for indole formation

Söderholm

Newton

Patrick

et al. 2020

Journal of Biological Chemistry

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In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGP synthase, IGPS) starts with a condensation step where the substrate’s carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation which restores the aromaticity of the phenyl. IGPS from Pseudomonas aeruginosa has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. This step was since the 1960s considered to be mechanistically essential based on studies of the IGPS:PRAI fusion protein from Escherichia coli. Here, we present the crystal structure of P. aeruginosa IGPS in complex with reduced CdRP, a non-reactive substrate analogue, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate (PAdRP) – with approximately 1000' lower rate of IGP formation than from the native substrate. We also show that E. coli IGPS, at even lower rate, can produce IGP from decarboxylated substrate. Our structure of P. aeruginosa IGPS has eight molecules in the asymmetric unit, out of which seven contain ligand, and one displays a previously unobserved conformation closer to the reactive state. One of the few non-conserved active-site residues, Phe201 in P. aeruginosa IGPS, is by mutagenesis demonstrated to be important for the higher turnover of this enzyme on both substrates. Our results demonstrate that, despite IGPS’s classification as a carboxylyase (i.e. decarboxylase), decarboxylation not a completely essential step in its catalysis.

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“…The canonical reaction mechanism ( 9 ) proceeds through the sequential steps of condensation, decarboxylation, and dehydration ( Fig. 1 B ).…”

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structure and kinetics of indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa: Decarboxylation is not essential for indole formation

Söderholm

Newton

Patrick

et al. 2020

Journal of Biological Chemistry

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“…The chemical reaction consists of a sequence of condensation, decarboxylation, and dehydration and is irreversible, because of both the formation of the pyrrole ring of the indole and the release of CO 2 . 1 The crystal structures of apo forms of the bifunctional enzyme IGPS/phosphoribosyl anthranilate isomerase from Escherichia coli (eIGPS:ePRAI) 2,3 as well as those of the monofunctional IGPS from the hyperthermophiles Sulfolobus solfataricus (sIGPS) 4,5 and Thermotoga maritima, 6 which optimally grow at ∼80 °C, have been determined at 2 Å resolution. IGPS has the ubiquitous (βα) 8 -barrel fold, with an N-terminal extension of ∼45 residues.…”

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confidence: 99%

“…Indole-3-glycerol phosphate synthase (IGPS) catalyzes the fifth reaction in the biosynthesis of tryptophan, whereby the enzyme facilitates a ring closure reaction of the substrate 1-( o -carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) to the product indole-3-glycerol phosphate (IGP) (Figure A). The chemical reaction consists of a sequence of condensation, decarboxylation, and dehydration and is irreversible, because of both the formation of the pyrrole ring of the indole and the release of CO 2 . The crystal structures of apo forms of the bifunctional enzyme IGPS/phosphoribosyl anthranilate isomerase from Escherichia coli (eIGPS:ePRAI) , as well as those of the monofunctional IGPS from the hyperthermophiles Sulfolobus solfataricus (sIGPS) , and Thermotoga maritima , which optimally grow at ∼80 °C, have been determined at 2 Å resolution.…”

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confidence: 99%

Kinetic Mechanism of Indole-3-glycerol Phosphate Synthase

et al. 2012

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The (βα)(8)-barrel enzyme indole-3-glycerol phosphate synthase (IGPS) catalyzes the multistep transformation of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) into indole-3-glycerol phosphate (IGP) in tryptophan biosynthesis. Mutagenesis data and crystal structure analysis of IGPS from Sulfolobus solfataricus (sIGPS) allowed for the formulation of a plausible chemical mechanism of the reaction, and molecular dynamics simulations suggested that flexibility of active site loops might be important for catalysis. Here we developed a method that uses extrinsic fluorophores attached to active site loops to connect the kinetic mechanism of sIGPS to structure and conformational motions. Specifically, we elucidated the kinetic mechanism of sIGPS and correlated individual steps in the mechanism to conformational motions of flexible loops. Pre-steady-state kinetic measurements of CdRP to IGP conversion monitoring changes in intrinsic tryptophan and IGP fluorescence provided a minimal three-step kinetic model in which fast substrate binding and chemical transformation are followed by slow product release. The role of sIGPS loop conformational motion during substrate binding and catalysis was examined via variants that were covalently labeled with fluorescent dyes at the N-terminal extension of the enzyme and mobile active site loop β1α1. Analysis of kinetic data monitoring dye fluorescence revealed a conformational change that follows substrate binding, suggesting an induced-fit-type binding mechanism for the substrate CdRP. Global fitting of all kinetic results obtained with wild-type sIGPS and the labeled variants was best accommodated by a four-step kinetic model. In this model, both the binding of CdRP and its on-enzyme conversion to IGP are accompanied by conformational transitions. The liberation of the product from the active site is the rate-limiting step of the overall reaction. Our results confirm the importance of flexible active loops for substrate binding and catalysis by sIGPS.

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Investigating the Roles of Active Site Residues in Mycobacterium tuberculosis Indole-3-glycerol Phosphate Synthase, a Potential Target for Antitubercular Agents

Konas,

Cho,

Thomas

et al. 2023

ACS Bio Med Chem Au

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Mycobacterium tuberculosis drug resistance is emerging and new drug targets are needed. Tryptophan biosynthesis is necessary for M. tuberculosis replication and virulence. Indole-3glycerol phosphate synthase (IGPS) catalyzes a step in M. tuberculosis tryptophan biosynthesis and has been suggested as a potential anti-infective target, but our understanding of this enzyme is limited. To aid in inhibitor design and gain a greater mechanistic picture of this enzyme, there is a need to understand the roles of active site amino acids in ligand binding and catalysis. In this work, we explored the roles of conserved active site amino acids Glu57, Lys59, Lys119, Glu168, and Glu219. Mutation of each to Ala results in loss of all detectable activity. The Glu57Gln, Lys59Arg, Lys119Arg, Glu168Gln, and Glu219Asp mutations result in large activity losses, while Glu219Gln has enhanced activity. Analysis of the enzymatic data yields the following main conclusions: (A) Lys119 is the likely catalytic acid in the CdRP ring closure step. (B) Glu168 stabilizes a charged reaction intermediate and may also be the catalytic base. (C) Glu57, Glu219, and Lys119 form a closely arranged triad in which Glu57 and Glu219 modulate the pK a of Lys119, and thus overall activity. This increased understanding of inter-and intramolecular interactions and demonstration of the highly coordinated nature of the M. tuberculosis IGPS active site provide new mechanistic information and guidance for future work with this potential new drug target.

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Biosynthesis of Compounds Containing an Indole Nucleus

Cited by 3 publications

References 175 publications

Structure and kinetics of indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa: Decarboxylation is not essential for indole formation

Structure and kinetics of indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa: Decarboxylation is not essential for indole formation

Kinetic Mechanism of Indole-3-glycerol Phosphate Synthase

Investigating the Roles of Active Site Residues in Mycobacterium tuberculosis Indole-3-glycerol Phosphate Synthase, a Potential Target for Antitubercular Agents

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