Chorismate mutase-prephenate dehydratase. Partial purification and properties of the enzyme from Salmonella typhimurium

Schmit, Joseph C.; Zalkin, Howard

doi:10.1021/bi00829a025

Cited by 62 publications

(32 citation statements)

References 15 publications

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“…In Aerobacter aerogenes, Salmonella typhimurium, and Escherichia coli, two forms of chorismate mutase have been found (3,12). One form is inhibited and repressed by phenylalanine and occurs in an aggregate with prephenate dehydratase.…”

Section: Discussionmentioning

confidence: 99%

Regulation of Aromatic Amino Acid Biosynthesis in Higher Plants

et al. 1972

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Etiolated mung bean seedlings were examined for chorismate mutase activity. Evidence for the occurrence of two forms of the enzyme (designated CM-1 and CM-2) was obtained by ammonium sulfate fractionation, anion exchange cellulose chromatography, and isoelectric focusing. The two forms showed distinctly different properties, as CM-1 was inhibited by phenylalanine and tyrosine and activated by tryptophan, but inhibition by phenylalanine and tyrosine was reversed by tryptophan. The other form, CM-2, was unaffected by any of the three aromatic amino acids. Isoelectric points of the two forms were CM-1, pH 4.6, and CM-2, pH 5.6. The molecular weights estimated by molecular sieving on Sephadex G-200 were CM-1, 50,000, and CM-2, 36,000.Chorismic acid has been established as an important intermediate in the biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan in microorganisms (5-7). By action of chorismate mutase, chorismate is converted to prephenate, the precursor of phenylalanine and tyrosine. Chorismate serves as a substrate for anthranilate synthetase, the first reaction in a branch pathway leading to the synthesis of tryptophan. Control of chorismate mutase activity would therefore be of prime importance in the regulation of phenylalanine and tyrosine biosynthesis. Multiple forms of chorismate mutase have been found in fungi, bacteria, and blue green algae. Certain of these forms appear to have regulatory properties, and their roles in regulating production of the two amino acids have been investigated in these microorganisms (2, 3, 8-14, 16, 17 MATERIALS AND METHODSPlant Material. Etiolated mung bean seedlings (Phaseolus aureus) were purchased from a local retail grocer. Such seedlings germinated in the laboratory and harvested 2 to 3 days after planting gave the same results as those obtained commercially.Preparation of Acetone Powders. Whole seedlings were added to a Waring Blendor and ground for 1 min with acetone precooled to -10 C. The resulting suspension was put on a Biichner funnel and washed with cold acetone until the filtrate was colorless. The acetone powder was air-dried and then stored in a desiccator at 4 C under nitrogen. Powders stored for 2 weeks retained at least 80% of chorismate mutase activity that was present at the time the powders were prepared. Those stored for 1 month possessed only one-half of their original activity.Preparation of Pulverized Frozen Material. Etiolated mung bean seedlings were frozen in liquid nitrogen and, while frozen, homogenized to a fine powder in a Waring Blendor. At least 60 sec at full speed were required to pulverize completely the frozen material. The powder was stored at -10 C until used for enzyme extraction. Powders stored at -10 C for 1 month retained at least 95% of their original chorismate mutase activity.Enzyme Preparation. Frozen powder was mixed with 20% by weight Polyclar AT and 50 mm potassium phosphate buffer. pH 6.8, 5 g fresh weight of powder per ml of buffer. The powder was allowed to thaw and then left ...

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Section: Discussionmentioning

confidence: 99%

Regulation of Aromatic Amino Acid Biosynthesis in Higher Plants

et al. 1972

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“…One unit of DAHP synthase is defined as the amount of protein catalyzing the appearance of 1 Mmol of DAHP (method A) or the disappearance of 1 Mmol of PEP (method B) per min. Chorismate mutase was assayed as described (24).…”

Section: Methodsmentioning

confidence: 99%

3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase from Carrot Root (Daucus carota) Is a Hysteretic Enzyme

Suzich¹,

Dean²,

Herrmann³

1985

Plant Physiol.

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Roots of carrots (Daucus carota) contain three activities of 3-deoxy-D-arabino-heptlsnte 7-phosphate (DAHP) synthase, the enzyme that catalyzes the first step of the shikimate pathway. The (10,19), Bacillus subtilis (13), Escherichia coli (17, 25), Salmonella typhimurium (11), Schizosaccharomyces pombe (2), and Brevibacterium flavum (29). The enzyme from cauliflower is the only plant DAHP synthase that has been substantially purified (14).Microorganisms employ a variety of mechanisms to control carbon flow through the shikimate pathway (8). While shikimate kinase appears to be the key allosteric control point of this metabolic route in B. subtilis (12) This paper shows that carrot roots contain three forms of DAHP synthase, enzymes I, II, and Ill; enzyme III is purified to electrophoretic homogeneity. This paper also presents a kinetic analysis that identifies enzyme III as hysteretic; hysteresis is affected by tryptophan, a feedback activator of the enzyme. MATERIALS AND METHODSReagents. PEP (4), erythrose-4-P (27), and DAHP (9) were synthesized and assayed as described previously (9, 25). All other reagents were obtained commercially at the highest purity available and were used without further purification.Enzyme Assays. DAHP synthase was assayed by either of two methods (25). In method A, the reaction mixture contained 0.75 Mmol of PEP, 0.3 Mmol of erythrose-4-P, 0.07 Mmol of L-tryptophan, 0.3 Mmol of MnCl2, and 100 ug of ovalbumin in a total volume of 0.1 ml of 50 mM bis-tris propane (pH 7.0). The reaction was initiated by adding 0.05 ml of appropriately diluted enzyme. The mixture was incubated for 20 min at 25C. The reaction was stopped with TCA. DAHP was determined after degradation with NaIO4 as the thiobarbiturate adduct. This endpoint assay was mainly used during the purification of the enzyme. In method B, 0.9 ml of appropriately diluted enzyme in 50 mm bis-tris propane (pH 7.0), containing 1 mM DTT and 150 Mm PEP were prewarmed to 25C. The reaction was started by addition of 100 Ml of 2.5 mm erythrose-4-P dissolved in the same buffer. The disappearance of PEP was continuously monitored by reading A232. This kinetic assay measures initial velocities and was used for most of the studies with the pure enzyme. One unit of DAHP synthase is defined as the amount of protein catalyzing the appearance of 1 Mmol of DAHP (method A) or the disappearance of 1 Mmol of PEP (method B) per min. Chorismate mutase was assayed as described (24).Protein Determination. Protein was determined by the method ofLowry et al. (15). Since DTT, bis-tris propane, and tryptophan interfere with this assay, protein was precipitated with 20% TCA and resuspended in 0.1 N NaOH, containing 0.2 M Na2CO3, prior to determination. During enzyme purification, protein in tryptophan containing buffers was determined by the method of Bradford (3) with lysozyme as the standard.Purification of DAHP Synthase Ill from Carrot Root (Dancus carota). All manipulations were carried out at 4C.Step . Fresh carrot roots were washed extensivel...

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“…This reaction sequence would be analogous to phosphorylation prior to decarboxylation by mevalonate 5-diphosphate decarboxylase (26) or sulfation prior to decarboxylation in curacin A biosynthesis (18). Alternatively, concomitant decarboxylation and dehydration without phosphorylation could take place as observed for prephenate dehydratase (9,47), but the latter reaction has a stronger driving force as a consequence of aromatization of the substrate. None of the proteins encoded by the ORFs of unknown function in P. syringae have sequences that are homologous with known decarboxylases.…”

Section: Discussionmentioning

confidence: 99%

Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

Kim

Metcalf

et al. 2012

Antimicrob Agents Chemother

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cFosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production in Pseudomonas syringae PB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that in Streptomyces fradiae and Streptomyces wedmorensis. The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome of P. syringae PB-5123 lacks a gene encoding the PnPy decarboxylase found in the Streptomyces strains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin in Pseudomonas aeruginosa from a fosmid encoding the fosfomycin biosynthetic cluster from P. syringae PB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent.

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Chorismate mutase-prephenate dehydratase. Partial purification and properties of the enzyme from Salmonella typhimurium

Cited by 62 publications

References 15 publications

Regulation of Aromatic Amino Acid Biosynthesis in Higher Plants

Regulation of Aromatic Amino Acid Biosynthesis in Higher Plants

3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase from Carrot Root (Daucus carota) Is a Hysteretic Enzyme

Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

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