Crystal Structure of Escherichia coli Enterobactin-specific Isochorismate Synthase (EntC) Bound to its Reaction Product Isochorismate: Implications for the Enzyme Mechanism and Differential Activity of Chorismate-utilizing Enzymes

Sridharan, Sridha; Howard, Nigel; Kerbarh, Olivier; Błaszczyk, M.; Abell, Chris; Blundell, Tom L.

doi:10.1016/j.jmb.2010.01.019

Cited by 30 publications

(88 citation statements)

References 34 publications

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“…This breakdown of either the substrate chorismic acid or of the product ADIC was also observed in the ligand-bound crystal structure of Serratia marcescens AS (PDB ID code 1I7Q) (2). The position and interactions of benzoate and pyruvate are very similar to those of the ADIC isoster isochorismate in the recently published isochorismate synthase EntC structure (28), showing that they serve as a good template for the enzyme/substrate complex (supplemental Fig. S5).…”

Section: Resultssupporting

confidence: 67%

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Ligand Binding Induces an Ammonia Channel in 2-Amino-2-desoxyisochorismate (ADIC) Synthase PhzE

Mavrodi

Thomashow

et al. 2011

Journal of Biological Chemistry

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PhzE utilizes chorismate and glutamine to synthesize 2-amino-2-desoxyisochorismate (ADIC) in the first step of phenazine biosynthesis. The PhzE monomer contains both a chorismateconverting menaquinone, siderophore, tryptophan biosynthesis (MST) and a type 1 glutamine amidotransferase (GATase1) domain connected by a 45-residue linker. We present here the crystal structure of PhzE from Burkholderia lata 383 in a ligandfree open and ligand-bound closed conformation at 2.9 and 2.1 Å resolution, respectively. PhzE arranges in an intertwined dimer such that the GATase1 domain of one chain provides NH 3 to the MST domain of the other. This quaternary structure was confirmed by small angle x-ray scattering. Binding of chorismic acid, which was found converted to benzoate and pyruvate in the MST active centers of the closed form, leads to structural rearrangements that establish an ammonia transport channel approximately 25 Å in length within each of the two MST/ GATase1 functional units of the dimer. The assignment of PhzE as an ADIC synthase was confirmed by mass spectrometric analysis of the product, which was also visualized at 1.9 Å resolution by trapping in crystals of an inactive mutant of PhzD, an isochorismatase that catalyzes the subsequent step in phenazine biosynthesis. Unlike in some of the related anthranilate synthases, no allosteric inhibition was observed in PhzE. This can be attributed to a tryptophan residue of the protein blocking the potential regulatory site. Additional electron density in the GATase1 active center was identified as zinc, and it was demonstrated that Zn 2؉ , Mn 2؉ , and Ni 2؉ reduce the activity of PhzE.Chorismic acid is the precursor for a large number of primary and secondary metabolites such as e.g. the aromatic amino acids, ubiquinone, folate, vitamin K, and the siderophores enterobactin and pyochelin. Because the production and utilization of chorismic acid are limited to prokaryotic microorganisms and plants, enzymes involved in chorismate metabolism are attractive targets for the development of biologically active compounds as exemplified by the herbicide glyphosate, which inhibits an enzyme of the shikimate pathway required for chorismate biosynthesis.Four chorismate-utilizing enzyme families are known (1). First, two unrelated types of chorismate mutases, AroH and AroQ, generate prephenate as a precursor of phenylalanine and tyrosine. A third family consists of the chorismate lyases, which cleave chorismic acid to p-hydroxybenzoate and pyruvate in the biosynthesis of ubiquinone. Finally, the fourth group comprises members of the menaquinone, siderophore, tryptophan (MST) 3 biosynthesis family, which utilize water or ammonia to perform nucleophilic substitution at the six-membered ring of chorismate with or without concomitant rearrangement of the double bond system in an Mg 2ϩ -dependent reaction (Fig. 1). Of these enzymes, isochorismate, 2-amino-2-desoxyisochorismate (ADIC), and 4-amino-4-desoxychorismate synthases release the isomerized product, whereas anthranilate (AS) ...

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Section: Resultssupporting

confidence: 67%

“…Similar refolding, but to a lesser extent, is also observed in AS (PDB ID codes 1I7Q and 1I7S) (2) and isochorismate synthase (PDB ID codes 3BZM and 3HWO) (28,29 , which leads to a peptide flip with respect to the open conformation (Fig. 3A).…”

supporting

confidence: 62%

Ligand Binding Induces an Ammonia Channel in 2-Amino-2-desoxyisochorismate (ADIC) Synthase PhzE

Mavrodi

Thomashow

et al. 2011

Journal of Biological Chemistry

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“…The crystal structure of EntC (46) with bound isochorismate indicates that the catalytic center contains a magnesium ion and the isochorismate product, but no cofactor, such as flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN), has been found to participate in EntC's catalytic reaction (28,46). There is no indication that EntC forms any inter-or intramolecular disulfide bonds or catalyzes any oxidation-reduction reaction (28,46). It has been shown that the function of the chorismate synthase in E. coli requires reduced FMN, but the NADPH:FMN oxidoreductase that provides the cofactor has not been identified (30).…”

Section: Fig 12mentioning

confidence: 99%

AhpC Is Required for Optimal Production of Enterobactin by Escherichia coli

Payne

2012

J Bacteriol

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Escherichia coli alkyl hydroperoxide reductase subunit C (AhpC) is a peroxiredoxin that detoxifies peroxides. Here we show an additional role for AhpC in cellular iron metabolism of E. coli. Deletion of ahpC resulted in reduced growth and reduced accumulation of iron by cells grown in low-iron media. Liquid chromatography-mass spectroscopy (LC-MS) analysis of culture supernatants showed that the ahpC mutant secreted much less enterobactin, the siderophore that chelates and transports ferric iron under iron-limiting conditions, than wild-type E. coli did. The ahpC mutant produced less 2,3-dihydroxybenzoate, the intermediate in the enterobactin biosynthesis pathway, and providing 2,3-dihydroxybenzoate restored wild-type growth of the ahpC mutant. These data indicated that the defect was in an early step in enterobactin biosynthesis. Providing additional copies of entC, which functions in the first dedicated step of enterobactin biosynthesis, but not of other enterobactin biosynthesis genes, suppressed the mutant phenotype. Additionally, providing either shikimate or a mixture of para-aminobenzoate, tryptophan, tyrosine, and phenylalanine, which, like enterobactin, are synthesized from the precursor chorismate, also suppressed the mutant phenotype. These data suggested that AhpC affected the activity of EntC or the availability of the chorismate substrate.

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“…Escherichia coli does not make SA, but it encodes two ICS enzymes EntC and MenF that are involved in siderophore biosynthesis (see ref. [17] and references therein). Engineering of transgenic Arabidopsis to express either a bifunctional SAS [18], or separate ICS and IPL enzymes [19], yielded plants that constitutively overproduced SA.…”

Section: Introductionmentioning

confidence: 99%

The biochemical properties of the two Arabidopsis thaliana isochorismate synthases

Macaulay

Heath

Ciulli

et al. 2017

Biochemical Journal

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The important plant hormone salicylic acid (SA; 2-hydroxybenzoic acid) regulates several key plant responses including, most notably, defence against pathogens. A key enzyme for SA biosynthesis is isochorismate synthase (ICS), which converts chorismate into isochorismate, and for which there are two genes in Arabidopsis thaliana. One (AtICS1) has been shown to be required for increased SA biosynthesis in response to pathogens and its expression can be stimulated throughout the leaf by virus infection and exogenous SA. The other (AtICS2) appears to be expressed constitutively, predominantly in the plant vasculature. Here, we characterise the enzymatic activity of both isozymes expressed as hexahistidine fusion proteins in Escherichia coli. We show for the first time that recombinant AtICS2 is enzymatically active. Both isozymes are Mg2+-dependent with similar temperature optima (ca. 33°C) and similar Km values for chorismate of 34.3 ± 3.7 and 28.8 ± 6.9 µM for ICS1 and ICS2, respectively, but reaction rates were greater for ICS1 than for ICS2, with respective values for Vmax of 63.5 ± 2.4 and 28.3 ± 2.0 nM s−1 and for kcat of 38.1 ± 1.5 and 17.0 ± 1.2 min−1. However, neither enzyme displayed isochorismate pyruvate lyase (IPL) activity, which would enable these proteins to act as bifunctional SA synthases, i.e. to convert chorismate into SA. These results show that although Arabidopsis has two functional ICS enzymes, it must possess one or more IPL enzymes to complete biosynthesis of SA starting from chorismate.

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Crystal Structure of Escherichia coli Enterobactin-specific Isochorismate Synthase (EntC) Bound to its Reaction Product Isochorismate: Implications for the Enzyme Mechanism and Differential Activity of Chorismate-utilizing Enzymes

Cited by 30 publications

References 34 publications

Ligand Binding Induces an Ammonia Channel in 2-Amino-2-desoxyisochorismate (ADIC) Synthase PhzE

Ligand Binding Induces an Ammonia Channel in 2-Amino-2-desoxyisochorismate (ADIC) Synthase PhzE

AhpC Is Required for Optimal Production of Enterobactin by Escherichia coli

The biochemical properties of the two Arabidopsis thaliana isochorismate synthases

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