Acetate activation in Methanosaeta thermophila: characterization of the key enzymes pyrophosphatase and acetyl-CoA synthetase

Abstract

The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PP(i)) and was only moderately inhibited by PP(i). The breakdown of PP(i) was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PP(i) (K(M) = 0.27 ± 0.05 mM) that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PP(i). However, it cannot be excluded that other PP(i)-dependent enzymes take advantage of the remaining PP(i) and contribute to the energy balance of the cell.

Figure 1

SDS-PAGE analysis of purified soluble pyrophosphatase (Mthe_0236) and AMP-dependent ACS (Mthe_1194). Enzymes were purified by Strep-tactin affinity chromatography. M: molecular mass marker (PAGE Ruler prestained protein ladder, Fermentas, St. Leon-Rot, Germany), lane 1: Mthe_0236 0.5 μg, lane 2: Mthe_1194 1 μg.

Figure 2

Activity measurement of the soluble type II pyrophosphatase (Mthe_0236). Assays contained 40 mM Tris-HCl pH 8 with 5 mM MgCl₂ and 1.25 μg enzyme/mL. (●) activity measurement after 5 min preincubation with 1 mM MnCl₂, (Δ) activity without preincubation, and (■) control without Mthe_0236.

Figure 3

Temperature stability of the AMP-dependent ACS Mthe_1194. (●) Incubation at 55°C, (▲) incubation at 75°C, and (■) incubation at 92°C. Enzyme activity was measured in the NADH consumption assay with auxiliary enzymes at 55°C.