Glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: expression of the g6pd gene and characterization of an extremely thermophilic enzyme.
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 degrees 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 degrees C the enzyme is the most thermoactive G6PD described.
Hansen
Thomas
T
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, D-24118, Kiel, Germany. thansen@ifam.uni-kiel.de
Schlichting
Bettina
B
Schönheit
Peter
P
D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005954:Glucosephosphate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D005954:Glucosephosphate Dehydrogenase