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 þ 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 31 . The enzyme also reduced NAD þ (apparent K m 12 mM, V max 12 U mg 31 ). The 1000-fold higher catalytic activity (k cat /K m ) with NADP þ over NAD þ defines the G6PD as NADP þ 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.

Intermediate instability at high temperature leads to low pathway efficiency for an in vitro reconstituted system of gluconeogenesis in Sulfolobus solfataricus

“…The cells were cultivated, harvested and disrupted as described in the respective publications (Ttx-PGI [35]; Ttx-PGK [9]; Ttx-HK [36]; Tma-G6PDH [37]; Sso-GDH [38]; Sso-PK [39]; Sso-GAPN [11]) or for the PGK and GAPDH as described above. The cellfree extracts obtained were subjected to heat precipitation at 80°C for 20 min and afterwards denatured proteins from the host were removed by centrifugation (16 000 g, 30 min, 4°C).…”

Cupin-Type Phosphoglucose Isomerases (Cupin-PGIs) Constitute a Novel Metal-Dependent PGI Family Representing a Convergent Line of PGI Evolution

“…The following controls were used. For His-tagged cPGIs, to correct for unspecific metal binding by the respective His tags as well as for H 2 O impurities, His-tagged glucose-6-phosphate dehydrogenase from T. maritima (23) and His-tagged PGI from P. aerophilum (25) were dialyzed and prepared as described above for the cPGIs, and the respective metal contents of the proteins were measured. The controls contained on average 0.02 Ni 2ϩ per enzyme molecule, whereas no iron could be detected in any of the control samples.…”

“…PGI activity (G6P 7 F6P) was determined in both directions. The formation of G6P and F6P was measured by using glucose-6-phosphate dehydrogenase from T. maritima (23) and mannitol-1-phosphate dehydrogenase from Klebsiella pneumoniae (41) as recently described (26). The following standard conditions were used: for PfcPGI, 80°C (pH 7.0) and 2 g of enzyme; for AfcPGI, 50°C or 70°C (pH 7.4) and 5 g of enzyme; for MmcPGI, 37°C (pH 7.4) and 20 g of enzyme; for StcPGI, 37°C (pH 7.2) and 33 g of enzyme; for EmcPGIA, 50°C (pH 7.4) and 80 g of enzyme.…”

Biological Hydrogen Production by Anaerobic Microorganisms

“…84 Whether the ED pathway may become dominant under certain conditions is not known. In the upper part of the ED pathway reducing equivalents are produced as NADPH, 85 which may only be used for anabolism, and thus are not available for hydrogen production. Several Thermotoga species are able to grow on complex carbohydrates, like cellulose or xylan and various endocellulases, cellobiohydrolases and xylanases have been purified and characterized.…”