ABSTRACT
The genome sequence of the non-sugar-assimilating mesophile Methanococcus maripaludis contains three genes encoding enzymes: a nonphosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR); all these enzymes are potentially capable of catalyzing glyceraldehyde-3-phosphate (G3P) metabolism. GAPOR, whose homologs have been found mainly in archaea, catalyzes the reduction of ferredoxin coupled with oxidation of G3P. GAPOR has previously been isolated and characterized only from a sugar-assimilating hyperthermophile, Pyrococcus furiosus (GAPORPf), and contains the rare metal tungsten as an irreplaceable cofactor. Active recombinant M. maripaludis GAPOR (GAPORMm) was purified from Escherichia coli grown in minimal medium containing 100 μM sodium molybdate. In contrast, GAPORMm obtained from cells grown in medium containing tungsten (W) and W and molybdenum (Mo) or in medium without added W and Mo did not display any activity. Activity and transcript analysis of putative G3P-metabolizing enzymes and corresponding genes were performed with M. maripaludis cultured under autotrophic conditions in chemically defined medium. The activity of GAPORMm was constitutive throughout the culture period and exceeded that of GAPDH at all time points. As GAPDH activity was detected in only the gluconeogenic direction and GAPN activity was completely absent, only GAPORMm catalyzes oxidation of G3P in M. maripaludis. Recombinant GAPORMm is posttranscriptionally regulated as it exhibits pronounced and irreversible substrate inhibition and is completely inhibited by 1 μM ATP. With support from flux balance analysis, it is concluded that the major physiological role of GAPORMm in M. maripaludis most likely involves only nonoptimal growth conditions.
In Vitro Reconstitution of an NADPH-Dependent Superoxide Reduction Pathway from Pyrococcus furiosus