Expression, Purification, and Characterisation of Dehydroquinate Synthase from Pyrococcus furiosus.

ABSTRACT: Dehydroquinate synthase (DHQS) catalyses the second step of the shikimate pathway to aromatic compounds. DHQS from the archaeal hyperthermophile Pyrococcus furiosus was insoluble when expressed in Escherichia coli but was partially solubilised when KCl was included in the cell lysis buffer. A purification procedure was developed, involving lysis by sonication at 30°C followed by a heat treatment at 70°C and anion exchange chromatography. Purified recombinant P. furiosus DHQS is a dimer with a subunit Mr of 37,397 (determined by electrospray ionisation mass spectrometry) and is active over broad pH and temperature ranges. The kinetic parameters are K(M) (3-deoxy-D-arabino-heptulosonate 7-phosphate) 3.7 μM and k(cat) 3.0 sec(-1) at 60°C and pH 6.8. EDTA inactivates the enzyme, and enzyme act

SUBMITTER: Negron L 

PROVIDER: S-EPMC3092513 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature