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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Overexpression, purification, and characterization of tyrosine-sensitive 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase from Escherichia coli.

An overexpression system (pCR105) for DAHP synthase (Tyr) was constructed by cloning the aroF gene at the NdeI site of the pET-22b(+) translation vector, a plasmid expression vector that contains the T7 lac promoter. The enzyme was overexpressed, purified to > 90% purity (by SDS-polyacrylamide gel electrophoresis), and characterized. The protein was overexpressed at a level of 58% the total soluble cell protein (based on enzymatic activities). About 244 mg of pure enzyme was obtained from a 2-liter cell culture. So far, this is the highest yield reported for the isozyme DAHP synthase (Tyr). The enzyme showed a bell-shaped pH-activity profile, with a pH optimum at pH 7.0-7.5 and pK values of 6.10 and 8.92. Inhibition of the enzyme by tyrosine was specific with 50% inhibition observed at 9 microM tyrosine, pH 7. 0. The specific activity of the enzyme increased with added metal and metal sensitivity increased with purity of the enzyme. Only substoichiometric amounts of Cu, Fe, and Zn were found in the pure enzyme and this result is consistent with sensitivity of the enzyme to added metal. Although treatment with EDTA inactivated the enzyme almost completely, the activity of the apoenzyme was restored to differing extents by a variety of metals including Mn2+, Cd2+, Co2+, Fe2+, Cu2+, Mg2+, and Zn2+. Both Fe2+ and Cu2+ only partially reactivated EDTA-treated enzyme. Reconstitution of EDTA-treated enzyme with either Cd2+ or Mn2+ gave 1 mol of metal per mole of enzyme monomer. KCN inactivated the enzyme to only 80% and added metals reactivated the CN-treated enzyme only to a small extent. These results confirm the importance of the metal in the enzymatic reaction.[1]


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