Purification and properties of tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.
The aroH gene of Escherichia coli, which encodes the tryptophan-sensitive 3-deoxy-D-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 Fe2+ 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%.
Ray
J M
JM
Department of Biology, University of Virginia, Charlottesville 22901.
Bauerle
R
R
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002412:Cations; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005246:Feedback; D015870:Gene Expression; D008667:Metalloproteins / Q000737:chemistry / Q000302:isolation & purification; D008670:Metals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013057:Spectrum Analysis; D014364:Tryptophan / Q000494:pharmacology
D002412:Cations; D008667:Metalloproteins; D008670:Metals; D014364:Tryptophan; D010723:3-Deoxy-7-Phosphoheptulonate Synthase