Purification and properties of the 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (phenylalanine sensitive) of Escherichia coli K12

Staub, Maria; Dénes, Géza

doi:10.1016/0005-2744(69)90228-9

Cited by 41 publications

(27 citation statements)

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“…As noted in earlier work with the other two DAHP synthase isoenzymes (24,28) synthases, DAHPS(Trp) was markedly sensitive to p-chloromercuribenzoate, which suggests a role for an essential cysteine(s) in the structure or activity of the enzyme. The recovery of an inactivating mutation at residue Cys-326, which is invariant in the three E. coli isoenzymes, after random mutagenesis of the aroH gene (21) supports this conclusion.…”

Section: Discussionsupporting

confidence: 59%

Purification and properties of tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli

Ray

Bauerle

1991

J Bacteriol

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The aroH gene of Escherichia coli, which encodes the tryptophan-sensitive 3-deoxy-n-arabino-heptulosonate-7-phosphate synthase isoenzyme of the common aromatic biosynthetic pathway, was cloned behind the tac promoter in expression plasmid pKK223-3. The enzyme was overexpressed, purified to homogeneity, and characterized. The native enzyme was found to be a dimeric metalloprotein containing 0.3 mol of iron per mol of subunit and variable amounts of zinc. The activity of the native enzyme was stimulated two-to threefold when assayed in the presence of Fe2+ ions. Pretreatment of the enzyme with Fe2+ also resulted in activation, accompanied by an equivalent increase in iron content. Treatment of the enzyme with chelating agents led to inactivation, which was fully reversed by the presence of Fe2+ in the assay mixture. The native enzyme exhibited a unique absorption profile, having a shoulder of absorbance on the aromatic band with a maximum around 350 nm and a broad, weak band with a maximum around 500 nm. Treatment of the enzyme with Fe2t enhanced the absorbance at 350 nm and eliminated the band at 500 nm. Treatment with reducing agents caused the disappearance of both bands and destabilized the enzyme. Feedback regulation of the activity of the enzyme was specific for tryptophan, with maximum inhibition at about 70%.In bacteria, a common aromatic pathway of seven steps leads to the synthesis of chorismic acid, the branch point precursor of the aromatic amino acids tryptophan, tyrosine, and phenylalanine, as well as of folic acid, ubiquinone, menaquinone, and enterochelin. The first committed step of this pathway is the condensation of erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP), yielding the phosphorylated 7-carbon keto sugar acid 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP). The enzyme that catalyzes this reaction is DAHP synthase (EC 4.1.2.15).In Escherichia coli there are three DAHP synthase isoenzymes, each specifically feedback regulated by one of the three aromatic amino acids (6). The Phe-sensitive [DAHP-S(Phe)] and Tyr-sensitive [DAHPS(Tyr)] isoenzymes, encoded by aroG and aroF, respectively, are responsible for essentially all of the DAHP synthase activity in the wild-type cell; the Trp-sensitive isoenzyme [DAHPS(Trp)], encoded by aroH, contributes only about 1% of the total activity (29). Molecular cloning and DNA sequencing of the aroF, aroG, and aroH genes (8,21,26) have revealed that the three polypeptides are nearly identical in size and have a high degree of sequence similarity. On this basis, it has been concluded that the three are homologs with a common evolutionary origin (19,21). DAHPS(Tyr) (1, 24) and DAHP-S(Phe) (17) from E. coli have been purified to homogeneity and extensively characterized; however, only a preliminary study of a partially purified preparation of DAHPS(Trp) has been reported (7). DAHPS(Phe) from E. coli is tetrameric in structure, while Bacterial strains and plasmids. E. coli AB3248 (aroF363 aroG365 aroH367 proA2 argE3 his4 thi lac gal-2 tsx-358) (31) and...

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Section: Discussionsupporting

confidence: 59%

Purification and properties of tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli

Ray

Bauerle

1991

J Bacteriol

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“…Previous experiments on KDO8P synthase showed the reaction is irreversible (7) and suggest the presence of an acyclic hemiketal phosphate intermediate in the catalytic mechanism of this enzyme (8,9). Also, experiments using radiolabeled H 2 18 O established that the reaction involves the nucleophilic attack of a water molecule on C-2 of PEP, followed by or in concert with the nucleophilic attack of C-3 of PEP on the C-1 of arabinose 5-phosphate. A similar mechanism can be envisioned for the E. coli DAHP synthase.…”

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confidence: 91%

“…Over the years, DAHP synthase has been isolated from a wide range of organisms ranging from bacteria to eukaryotes (1)(2)(3)(4)(5). Escherichia coli harbors three DAHP synthase isozymes, each subject to feedback inhibition by one of the three aromatic amino acids (tyrosine, phenylalanine, and tryptophan, respectively).…”

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confidence: 99%

“…From the first time DAHP synthase was isolated from E. coli in 1969 (2), there has been an ongoing controversy in the literature regarding the nature of the metal ion at the active site of this enzyme. A variety of metal ions have been proposed to reside at the active site of DAHP synthase under physiological conditions.…”

mentioning

confidence: 99%

“…A variety of metal ions have been proposed to reside at the active site of DAHP synthase under physiological conditions. They include cobalt (2,11), iron (4,12), or copper (13). Today, after more than 30 years of research, the nature of the physiological metal ion at the active site of DAHP synthase remains an open question.…”

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confidence: 99%

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Insights into the Mechanism of 3-Deoxy-D-arabino-heptulosonate 7-Phosphate Synthase (Phe) from Escherichia coli Using a Transient Kinetic Analysis

Furdui

Zhou

Woodard

et al. 2004

Journal of Biological Chemistry

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Escherichia coli phenylalanine-sensitive 3-deoxy-arabino-heptulosonate 7-phosphate synthase (DAHP synthase) catalyzes the net aldol condensation of phosphoenolpyruvate and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate and inorganic phosphate. For the first time, the presteady-state kinetic analysis of the Phe-sensitive DAHP synthase from E. coli is reported. The steady-state and presteadystate kinetic parameters of the DAHP synthase reconstituted with Mn(II), Cu(II), and Zn(II) were compared. These studies showed the following: 1) product release is rate-limiting for all of the three metal ions studied under physiologically relevant conditions; 2) concentration of the active sites of the metal-containing DAHP synthase is increasing from Mn-(30%) to Zn-(52%) and to Cu-DAHP synthase (88%); 3) rate constant for product formation is higher in Mn-(130 -200 s ؊1 ) than Cu-(55 s ؊1 ) and Zn-DAHP synthase (6.8 s ؊1 ); and 4) steady-state rate (rate constant for product release) is higher for the Mn-(70 s ؊1 ) than for Cu-(5.6 s ؊1 ) and Zn-DAHP synthase (1.8 s ؊1 ). In addition, an examination of the reaction kinetics at lower pH reveals that for Cu-DAHP synthase, product release is no longer rate-limiting, whereas the Mn-and Zn-DAHP synthase show a slower rate of product formation, suggesting that the intermediate formation becomes rate-limiting in product formation. Also, a deuterium-isotope effect on the burst rate constant of product formation for Mn-DAHP synthase was observed at pH 6.0. This supports the hypothesis that the role of metal ion in E. coli DAHP synthase is to position the amino acids with the appropriate geometry required to coordinate and activate the water molecule.The first committed step in the biosynthesis of shikimic acid, which ultimately leads to the synthesis of aromatic amino acids, is catalyzed by 3-deoxy-arabino-heptulosonate 7-phosphate (DAHP) 1 synthase. The biosynthetic pathway of aromatic amino acids is absent in mammals, rendering the enzyme an attractive molecular target for antibiotic design. A putative inhibitor for the enzyme would inhibit bacterial growth while leaving the host organism unaffected.Over the years, DAHP synthase has been isolated from a wide range of organisms ranging from bacteria to eukaryotes (1-5). Escherichia coli harbors three DAHP synthase isozymes, each subject to feedback inhibition by one of the three aromatic amino acids (tyrosine, phenylalanine, and tryptophan, respectively). In the present study, we have focused on the phenylalanine-sensitive DAHP synthase encoded by E. coli aroG gene. The enzyme catalyzes the net aldol condensation of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form DAHP and inorganic phosphate (P i ) (Scheme 1).The degree of sequence homology between the E. coli DAHP synthase and the 3-deoxy-D-manno-2-octulosonic acid-8-phosphate (KDO8P synthase) and the conserved active site residues between the two enzymes allow us to extrapolate the mechanistic findings from one system to the other (6). ...

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Is allostery a fuzzy concept?

Morea,

Angelucci,

Bellelli

2024

FEBS Open Bio

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Allostery is an important property of biological macromolecules which regulates diverse biological functions such as catalysis, signal transduction, transport, and molecular recognition. However, the concept was expressed using two different definitions by J. Monod and, over time, more have been added by different authors, making it fuzzy. Here, we reviewed the different meanings of allostery in the current literature and found that it has been used to indicate that the function of a protein is regulated by heterotropic ligands, and/or that the binding of ligands and substrates presents homotropic positive or negative cooperativity, whatever the hypothesized or demonstrated reaction mechanism might be. Thus, proteins defined to be allosteric include not only those that obey the two‐state concerted model, but also those that obey different reaction mechanisms such as ligand‐induced fit, possibly coupled to sequential structure changes, and ligand‐linked dissociation‐association. Since each reaction mechanism requires its own mathematical description and is defined by it, there are many possible ‘allosteries’. This lack of clarity is made even fuzzier by the fact that the reaction mechanism is often assigned imprecisely and/or implicitly in the absence of the necessary experimental evidence. In this review, we examine a list of proteins that have been defined to be allosteric and attempt to assign a reaction mechanism to as many as possible.

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Purification and properties of the 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (phenylalanine sensitive) of Escherichia coli K12

Cited by 41 publications

References 13 publications

Purification and properties of tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli

Purification and properties of tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli

Insights into the Mechanism of 3-Deoxy-D-arabino-heptulosonate 7-Phosphate Synthase (Phe) from Escherichia coli Using a Transient Kinetic Analysis

Is allostery a fuzzy concept?

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