Enzymatic degradation of cyclic 2,3-diphosphoglycerate to 2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum

Abstract

2,3-Diphosphoglycerate (2,3-DPG) has been found to be the product of the enzymatic degradation of cyclic 2,3-diphosphoglycerate (cDPG) in the archaebacterium Methanobacterium thermoautotrophicum delta H. Although 2,3-DPG has not previously been detected as a major soluble component of M. thermoautotrophicum, large pools accumulated at an incubation temperature of 50 degrees C (below the optimum growth temperature of 62 degrees C). Under these conditions, cellular activity was significantly decreased; a return of the culture to the optimum growth temperature restored the 2,3-DPG pool back to original low levels and caused steady-state cDPG levels to increase again. While 13CO2-pulse/12CO2-chase experiments at 50 degrees C showed that the cDPG turned over, the appearance of 2,3-DPG at NMR-visible concentrations required at least 10 h. Production of 2,3-DPG in vivo was prevented by exposure of the cells to O2. The enzyme responsible for this hydrolysis of cDPG was purified by affinity chromatography and appears to be a 33-kDa protein. Activity was detected in the presence of oxygen and was enhanced by a solution of 1 M KCl, 25 mM MgCl2, and dithiothreitol. Both Km and Vmax have been determined at 37 degrees C; kinetics also indicate that in vitro the product, 2,3-DPG, is an inhibitor of cDPG hydrolysis. These findings are discussed in view of a proposed role for cDPG in methanogens.

Halotolerance of Methanobacterium thermoautotrophicum delta H and Marburg

“…Cells of M. thermoautotrophicum AH were routinely grown in 250-ml serum bottles containing 50 ml of medium of the composition described earlier (42). Detailed conditions for cell growth have been provided previously (34). Basal medium includes 0.01 M NaCl, with other species contributing to a total Na+ concentration equal to 0.04 M. In the analysis of the effect of NaCl (from 0.01 to 0.6 M) on growth and methanogenesis, only the NaCl rather than the total Na+ concentration is given, since levels of the other Na+ salts remain constant.…”

“…The total Na+ concentration in the basal medium including the NaCl was 21.6 mM. To examine the effect of NaCl on cells of strain Marburg, a culture grown in basal medium was sequentially transferred into medium with increasing amounts (0.26 to 1.0 M) of external NaCl; the effect of yeast extract (0.1%), glycine (16.8 (19,34,36,41). Using ratios of the amount of cDPG to each other solute, determined from the natural abundance '3C spectrum, the micromoles of solutes in each extract were estimated (comparing all CH2 intensities in other molecules with the CH2 intensity of cDPG and comparing CH intensities with the CH intensity of cDPG).…”

Organic compatible solutes of halotolerant and halophilic microorganisms

“…However, in Methanobacter thermoautotrophicus , the K + -activated cDGPS appears irreversible (and membrane-bound), suggesting it may have a different role [105]. In soluble cell extracts, hydrolysis of cDPG in the presence of ADP and Pi could not generate ATP [106]. However, a membrane bound hydrolase that is inhibited by K + and Pi has also been identified [105].…”

Activation and thermostabilization effects of cyclic 2,3-diphosphoglycerate on enzymes from the hyperthermophilic Methanopyrus kandleri

“…Functions of cDPG in phosphate storage (Seeley and Fahrney 1984), energy storage (Gorris et al 1990;van Alebeek et al 1991), and biosynthesis (Evans et al 1986;Gorkovenko and Roberts 1993) have also been discussed. cDPG is synthesized from 2,3-bisphosphoglycerate in an ATP-dependent reaction (Lehmacher et al 1990;Van Alebeek et al 1994a) and is converted back to 2,3-diphosphoglycerate via a specific hydrolase (Sastry et al 1992;Van Alebeek et al 1994b). In methanogens, 2,3-bisphosphoglycerate, which is an intermediate in gluconeogenesis, is synthesized from pyruvate via phosphoenolpyruvate and 2-phosphoglycerate involving phosphoenolpyruvate synthetase and 2-phosphoglycerate kinase (Van Alebeek et al 1994a).…”