Expression, purification, and characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Pyrococcus furiosus.
Abstract The enzyme 3-deoxy- d - arabino -heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus has been expressed in Escherichia coli . The expressed protein was insoluble but was partially solubilized as a dimer by the inclusion of 200 mM KCl in the cell lysis buffer. An effective two step purification procedure has been developed. The first step resulted in a high degree of purification and involved lysis by sonication at approximately 40 °C followed by a heat treatment at 70 °C. A continuous assay measuring the loss of PEP at 232 nm at elevated temperatures was also developed. Temperature, pH, and divalent metal ions all had an effect on the extinction coefficient of PEP. Purified recombinant P. furiosus DAH7PS is a dimer with a subunit M r of 29,226 (determined by ESMS), shows resistance to denaturation by SDS, has activity over a broad pH range, and has an activation energy of 88 kJ mol −1 . The kinetic parameters are K m (PEP) 120 μM, K m (E4P) 28 μM, and k cat 1.5 s −1 , at 60 °C and pH 6.8. DAH7PS is not inhibited by phenylalanine, tyrosine, or tryptophan. EDTA inactivates the enzyme and enzyme activity is restored by a wide range of divalent metal ions including (in order of decreasing effectiveness): Zn 2+ , Cd 2+ , Mn 2+ , Co 2+ , Ni 2+ , Ca 2+ , Hg 2+ , and Cu 2+ . This detailed characterization of the DAH7PS from P. furiosus raises the possibility that the subfamily Iβ DAH7PS enzymes are metal ion dependent, contrary to previous predictions.