An Essential GTPase, Der, Containing Double GTP-binding Domains from Escherichia coli and Thermotoga maritima

A gene encoding a putative GTPase containing two tandemly repeated GTP-binding domains from a hyperthermophilic bacterium, Thermotoga maritima, was cloned and expressed in Escherichia coli. The gene (TM1446) termed der is highly conserved in Eubacteria including E. coli. The purified der product (Tm-Der) has GTPase activity but no ATPase activity. GTP, GDP, and dGTP but not GMP, ATP, CTP, and UTP compete for GTP binding to Tm-Der. An optimal condition for the GTPase assay was determined to be pH 7.5 in 400 mM KCl and 5 mM MgCl₂ at 70 °C, where K_m, V_max, and k _cat values were determined to be 110 μM, 3.46 μM/min, and 0.87 min^-1, respectively. A der deletion strain of E. coli was constructed by replacing the der gene (originally annotated yfgK) with a kanamycin resistance gene. The deletion strain was found to form colonies only if the cells harbored a plasmid containing der, indicating that der is essential for E. coli growth.