Biochemical and biophysical characterisation yields insights into the mechanism of a Cd/Zn transporting ATPase purified from the hyperaccumulator plant Thlaspi caerulescens

TcHMA4 (GenBank no. AJ567384), a Cd/Zn transporting ATPase of the P-1B-type (=CPx-type) was isolated and purified from roots of the Cd/Zn hyperaccumulator Thlaspi caerulescens. Optimisation of the purification protocol, based on binding of the natural C-terminal His-tag of the protein to a Ni-IDA metal affinity column, yielded pure, active TcHMA4 in quantities sufficient for its biochemical and biophysical characterisation with various techniques. TcHMA4 showed activity with Cu(2+), Zn(2+) and Cd(2+) under various concentrations (tested from 30 nM to 10 mu M), and all three metal ions activated the ATPase at a concentration of 0.3 mu M. Notably, the enzyme worked best at rather high temperatures, with an activity optimum at 42 degrees C. Arrhenius plots yielded interesting differences in activation energy. In the presence of zinc it remained constant (E-A=38 kJ.mol(-1)) over the whole concentration range while it increased from 17 to 42 kJ.mol(-1) with rising copper concentration and decreased from 39 to 23 kJ.mol(-1) with rising cadmium concentration. According to EXAFS the TcHMA4 appeared to bind Cd(2+) mainly by thiolate sulphur from cysteine, and not by imidazole nitrogen from histidine. (C) 2011 Elsevier B.V. All rights reserved.