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

摘要

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 80degreesC) 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 92degreesC the enzyme is the most thermoactive G6PD described. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. [References: 20]