Modification of a catalytically important residue of indoleglycerol‐phosphate synthase from <i>Escherichia coli</i>

Eberhard, Marc O.; Kirschner, Kasper

doi:10.1016/0014-5793(89)80225-x

Cited by 11 publications

(8 citation statements)

References 12 publications

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“…5F). Previously, the role of Arg-182 was attributed to stabilization of the negatively charged carboxylate of CdRP (23). From MD structures, it is evident that the catalytically crucial Arg-182 plays an important role in the stabilization of the negatively charged phosphate moiety of IGPS.…”

Section: Lys-110-nhmentioning

confidence: 99%

Molecular dynamics studies of ground state and intermediate of the hyperthermophilic indole-3-glycerol phosphate synthase

Mazumder-Shivakumar

Bruice

2004

Proc. Natl. Acad. Sci. U.S.A.

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Indole-3-glycerol phosphate synthase catalyzes the terminal ring closure step in tryptophan biosynthesis. In this paper, we compare the results from molecular dynamics (MD) simulations of enzymebound substrate at 298, 333, 363, and 385 K and the enzyme-bound intermediate at 385 K, solvated in TIP3P water box with a CHARMM force field. Results from MD simulations agree with experimental studies supporting the observation that Lys-110 is the general acid. Based on its location in the active site during the MD simulations, Glu-210 warrants classification as the general base instead of the previously proposed Glu-159. We find that the relative population of the reactive enzyme-substrate Michaelis conformers [near attack conformers (NACs)] with temperature correlates well (correlation coefficient of 0.96) with the relative activity of this thermophilic enzyme. At higher temperature, the enzyme-substrate electrostatic interaction favors the binding of the substrate in NAC conformation, whereas, at lower temperature, the substrate is distorted and bound in a nonreactive conformation. This change is reflected in the Ϸ1,100-fold increase in population of NACs at 385 K relative to 298 K. The easily determined population of NACs at given temperature tells much about the thermophilic property of the enzyme. Thus, the hyperthermophilic enzyme has evolved to have optimum activity at high temperatures, and, with lowering of the temperature, the electrostatic interaction at the active site is enhanced and the structure is deformed. This model can be regarded as a general explanation for the activity of hyperthermophilic enzymes.T he reduced flexibility of hyperthermophilic enzymes is responsible for their stability at high temperature (1, 2). Hyperthermophilic enzymes do not denature at high temperatures because of an abundance of electrostatic interactions, such as increased number of salt bridges. The question commonly asked is how thermophilic enzyme achieves the balance between the need for increased stability at high temperatures and the need for sufficient flexibility to function properly. We address the problem of how a thermophilic enzyme achieves greater rate enhancement at higher temperature and the role of the ground state conformers in doing so.The enzyme chosen for this study is the monofunctional hyperthermophilic indole-3-glycerol phosphate (IGP) synthase (IGPS) from the archaeon Sulfolobus solfataricus and is a member of the family of enzymes having (␤␣) 8 -fold barrel, commonly known as the TIM barrel (3). The natural habitat of S. solfataricus is sulfurous mud pools with temperatures Ϸ385 K. The IGPS is the terminal enzyme in the tryptophan biosynthesis pathway that catalyzes the ring closure of the substrate 1-(ocarboxyphenylamino) 1-deoxyribulose 5-phosphate (CdRP) to form the product IGP (Scheme 1) (4). Based on the available crystal structures and by modeling the intermediate in the enzyme active site (In), Kirschner and coworkers (5) provided useful insights into the catalytic mechanism of IGPS. The gener...

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Section: Lys-110-nhmentioning

confidence: 99%

Molecular dynamics studies of ground state and intermediate of the hyperthermophilic indole-3-glycerol phosphate synthase

Mazumder-Shivakumar

Bruice

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…An alternative hypothesis is that another active-site residue, such as Lys-53, also acts as a general acid, likely during the dehydration step. Indeed, site-directed mutagenesis of Escherichia coli IGPS demonstrated that Lys-53 (Lys-55 in E. coli IGPS) is highly important to IGPS catalysis (13). Lys-53 may also play roles in substrate binding and structural rearrangements occurring during catalysis.…”

mentioning

confidence: 99%

Functional Identification of the General Acid and Base in the Dehydration Step of Indole-3-glycerol Phosphate Synthase Catalysis

Zaccardi

Yezdimer

Boehr

2013

Journal of Biological Chemistry

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Background: Bacteria require the enzyme indole-3-glycerol phosphate synthase for the production of Trp. Results: Glu-51 and Lys-53 are identified as the base and acid acting in the dehydration step of enzyme catalysis. Conclusion: Ring closure and dehydration steps are catalyzed by distinct active-site surfaces. Significance: Enzyme inhibitors targeted against these active-site surfaces may serve as novel antibiotics.

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“…The conserved residues Lys53, Lys110, Glu159 and Asn180 (SsIGPS numbering) have been demonstrated essential for activity (corresponding to Lys63, Lys123, Glu172 and Asn194 in PaIGPS, Fig. 2) (11,12) and based on the SsIGPS-ligand structures, Lys110 was suggested to act as catalytic acid for both condensation and dehydration, while Glu159 was suggested to act as catalytic base in the dehydration step (7). Later, Zaccardi et al argued that it is unlikely for Lys110 to act as catalytic acid in both reaction steps since there is no clear mechanism for its reprotonation.…”

Section: Introductionmentioning

confidence: 99%

“…The suggested catalytic residues (K123 and E172) according to Hennig et al(7) are marked with black asterisks and the alternative catalytic residues (K63 and E61) suggested by Zaccardi et al(13) by gray asterisks. Essential residues(11,12) are marked with red rings. The residue targeted for mutagenesis in this study is marked in a yellow box.…”

mentioning

confidence: 99%

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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Modification of a catalytically important residue of indoleglycerol‐phosphate synthase from Escherichia coli

Cited by 11 publications

References 12 publications

Molecular dynamics studies of ground state and intermediate of the hyperthermophilic indole-3-glycerol phosphate synthase

Molecular dynamics studies of ground state and intermediate of the hyperthermophilic indole-3-glycerol phosphate synthase

Functional Identification of the General Acid and Base in the Dehydration Step of Indole-3-glycerol Phosphate Synthase Catalysis

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

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