Biochemical and biophysical characterisation yields insights into the mechanism of TcHMA4, a Cd/Zn transporting ATPase purified from the hyperaccumulator plant Thlaspi caerulescens

Abstract

TcHMA4 (GenBank no. AJ567384), a Cd/Zn transporting ATPase of the P1B-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 μM), and all three metal ions activated the ATPase at a concentration of 0.3 μM. Notably, the enzyme worked best at rather high temperatures, with an activity optimum at 42 °C. Arrhenius plots yielded interesting differences in activation energy. In the presence of zinc it remained constant (EA=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.