Purification of the NADP+: F420oxidoreductase ofMethanosphaera stadtmanae

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Abstract
Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420oxidoreductase activity. The NADP+:F420oxidoreductase, the enzyme which catalyses the electron transfer between NADP+and F420in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420reduction was 6.0, and 8.5 for NADP+reduction. During the purification process, it was noted that precipitation with (NH4)2SO4increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of α, β, and γ subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Kmvalues were 370 µM for NADP+, 142 µM for NADPH, 62.5 µM for F420, and 7.7 µM for F420H2. These values were different from the Kmvalues observed in the cell-free extract.Key words: methanogen, NADP:F420oxidoreductase, NADP reductase, F420, NADP+.