Glyceraldehyde-3-phosphate ferredoxin oxidoreductase from Methanococcus maripaludis

Fujirebio Inc., Research & Development Division, 51 Komiya-cho, Hachioji-shi, Tokyo 192-0031, Japan

^* To whom correspondence should be addressed. Email: js-patrik{at}fujirebio.co.jp.

	Abstract

The genome sequence of the non-sugar assimilating mesophile, Methanococcus maripaludis, contains three genes encoding enzymes (GAPN, GAPDH, GAPOR) potentially capable of catalyzing glyceraldehyde-3-phosphate (G3P) metabolism, including a homolog of Pyrococcus furiosus G3P:ferredoxin-oxidoreductase (mmGAPOR). GAPOR, whose homologs have been mainly found in archaea, catalyzes the reduction of ferredoxin coupled with oxidation of G3P. GAPOR has previously only been isolated and characterized from a sugar assimilating hyper-thermophile, Pyrococcus furiosus (pfGAPOR), and contains the rare metal tungsten as an irreplaceable cofactor.

Active recombinant GAPOR was purified from Escherichia coli grown in minimal medium containing 100 µM sodium molybdate. In contrast, mmGAPOR obtained from cells grown in W-, W and Mo-containing media, and medium without added W and Mo did not display any activity. Activity and transcript analysis of putative G3P-metabolizing enzymes and corresponding genes was performed with M. maripaludis cultured under autotrophic conditions in chemically defined medium. The activity of GAPOR was constitutive throughout the culture period and exceeded that of GAPDH at all time-points. As GAPDH activity only was detected in the gluconeogenic direction, whilst GAPN-activity was completely absent, only mmGAPOR catalyzes oxidation of G3P in M. maripaludis. Recombinant mmGAPOR is post-transcriptionally 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 GAPOR in M. maripaludis most likely will be under non-optimal growth conditions.