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-bind ing domains from a hyperthermophilic bacterium, Thermotoga maritima, was cl oned and expressed in Escherichia coli. The gene (TM1446) termed der is hig hly conserved in Eubacteria including E. coli. The purified der product (Tm -Der) has GTPase activity but no ATPase activity. GTP, GDP, and dGTP but no t GMP, ATP, CTP, and UTP compete for GTP binding to Tm-Der. An optimal cond ition for the GTPase assay was determined to be pH 7.5 in 400 mm KCI and 5 mM MgCl2 at 70 degreesC, where K-m, V-max, and k(cat) values were determine d to be 110 mum, 3.46 mum/min, and 0.87 min(-1), respectively. A der deleti on strain of E. coli was constructed by replacing the der gene (originally annotated yfgK) with a kanamycin resistance gene. The deletion strain was f ound to form colonies only if the cells harbored a plasmid containing der, indicating that der is essential for E. coli growth.