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

Abstract

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...

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

“…[1-14 C]PEP Synthesis-Radiolabeled PEP was enzymatically synthesized from [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]pyruvate by coupling the pyruvate phosphate dikinase reaction to inorganic pyrophosphatase reaction. Pyruvate phosphate dikinase was a generous gift from Prof. Dunaway-Mariano at the University of New Mexico.…”

“…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.…”

The Gene Expression and Enzyme Activity of Plant 3-Deoxy-D-Manno-2-Octulosonic Acid-8-Phosphate Synthase Are Preferentially Associated with Cell Division in a Cell Cycle-Dependent Manner

“…Because the assay used for enzymatic activity monitors the formation of 3-deoxy-ald-2-ulosonic acids (Ray, 1980), it was necessary to show that the recombinant tomato Kdo-8-P synthase was specific for Ara-5-P. d-Erythrose-4-P and d-Rib-5-P were assayed as substrates with protein extracts from the transformed S. enterica strains at 42°C (Table I). Using d-erythrose-4-P, the value of residual 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase activity in AG701i50-pLEKdoS cell extracts was not significantly different from that of the residual Kdo-8-P synthase activity in AG701i50 or AG701i50-pBluescript.…”

“…The cell debris was discarded after a 10-min centrifugation at 25,000g, and the resulting supernatant was desalted by G25-Sephadex column chromatography. The specific enzymatic activity of Kdo-8-P synthase was determined by the thiobarbituric acid assay as described by Ray (1980) using crude bacterial extracts of the different S. enterica strains or tomato plant tissue extracts.…”

(De)regulation of key enzyme steps in the shikimate pathway and phenylalanine-specific pathway of the actinomycete Amycolatopsis methanolica

“…DAHP synthase enzymes are present either as a monofunctional di-or tetrameric protein, e.g. in Escherichia coli (Ray & Bauerle, 1991;McCandliss et al, 1978;Schoner & Herrmann, 1976), or as a bifunctional protein exhibiting both DAHP synthase and CM activities, e.g. in Brevibacterium flavum (Sugimoto & Shiio, 1980) and in A. methanolica (Euverink et al, 1995a).…”