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Enzyme Research
Volume 2011 (2011), Article ID 134893, 10 pages
Research Article

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

1Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand
2Institute of Molecular Biosciences, Massey University, Palmerston North 4442, New Zealand
3Department of Chemistry, Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand

Received 6 October 2010; Revised 28 January 2011; Accepted 8 February 2011

Academic Editor: Vasu D. Appanna

Copyright © 2011 Leonardo Negron et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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 30C followed by a heat treatment at 70C 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 KM (3-deoxy-D-arabino-heptulosonate 7-phosphate) 3.7 μM and kcat 3.0 sec-1 at 60C and pH 6.8. EDTA inactivates the enzyme, and enzyme activity is restored by several divalent metal ions including (in order of decreasing effectiveness) Cd2+, Co2+, Zn2+, and Mn2+. High activity of a DHQS in the presence of Cd2+ has not been reported for enzymes from other sources, and may be related to the bioavailability of Cd2+ for P. furiosus. This study is the first biochemical characterisation of a DHQS from a thermophilic source. Furthermore, the characterisation of this hyperthermophilic enzyme was carried out at elevated temperatures using an enzyme-coupled assay.