Glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: expression of the g6pd gene and characterization of an extremely thermophilic enzyme

Abstract

The gene (open reading frame Tm1155, g6pd) encoding glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) of the hyperthermophilic bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 95 kDa composed of 60-kDa subunits. Rate dependence (at 80°C) on glucose-6-phosphate and NADP + followed Michaelis–Menten kinetics with apparent K m values of 0.15 mM and 0.03 mM, respectively; apparent V max values were about 20 U mg −1. The enzyme also reduced NAD + (apparent K m 12 mM, V max 12 U mg −1). The 1000-fold higher catalytic activity ( k cat/ K m) with NADP + over NAD + defines the G6PD as NADP + specific in vivo. G6PD activity was competitively inhibited by NADPH with a K i value of 0.11 mM. With a temperature optimum of 92°C the enzyme is the most thermoactive G6PD described.