The extremely thermophilic eubacterium, Thermotoga maritima, contains a novel iron-hydrogenase whose cellular activity is dependent upon tungsten

Characterization of Di-myo-Inositol-1,1(prm1)-Phosphate in the Hyperthermophilic Bacterium Thermotoga maritima

“…Any significant insight into this problem obviously has many biotechnological ramifications. While most of the enzymes that have been purified from hyperthermophiles exhibit maximal catalytic activity above 90ЊC in vitro (1,2), the assays typically used are of short duration, and many of the pure proteins are not stable over prolonged periods at the optimum growth temperature of the organism from which they were obtained (4,15,21). This observation has led to the notion that these organisms may contain solutes that play a general "thermoprotectant" role to stabilize some, if not all, of their cytoplasmic proteins.…”

“…T. maritima (DSM 3109) was grown with glucose as the carbon source in a 600-liter fermentor at 80ЊC with the medium previously described (15), except that (NH 4 ) 2 CO 3 (12 mM), KCl (26.8 mM), CaCl 2 (0.45 mM), and yeast extract (0.25%, wt/vol) were at the indicated concentrations and NaI, H 3 BO 4 , SrCl 2 , and KI were omitted. After the medium was sterilized and cooled to 80ЊC, dipotassium phosphate (3.7 mM) was added and cysteine hydrochloride (2.3 mM) was used as the reductant.…”

“…In order to investigate the properties of DIP, a large-scale purification procedure was devised. The cytoplasmic fraction was prepared from 470 g (wet weight) of frozen T. maritima cells as previously described (15), except that the buffer was 50 mM Tris HCl (pH 8.0). This was applied to a column (10 by 20 cm) of DEAE Sepharose (Pharmacia LKB, Piscataway, N.J.) equilibrated with the same buffer.…”

“…The efficacy of DIP in minimizing protein denaturation at extreme temperatures was investigated by using two enzymes previously purified from T. maritima: pyruvate ferredoxin oxidoreductase (POR) (4) and hydrogenase (15). POR catalyzes the oxidative decarboxylation of pyruvate to acetyl coenzyme A and the electrons are transferred to the redox protein ferredoxin for ultimate disposal as H 2 , which is catalyzed by the hydrogenase (1).…”

“…POR catalyzes the oxidative decarboxylation of pyruvate to acetyl coenzyme A and the electrons are transferred to the redox protein ferredoxin for ultimate disposal as H 2 , which is catalyzed by the hydrogenase (1). Both are iron-sulfur-containing proteins and both are inactivated by O 2 (4,15). The pure enzymes were incubated at 90ЊC under anaerobic conditions with and without DIP (at pH 8.0).…”