Abstract
Analysis of genome sequence data from the methanogenic archaeon Methanosarcina mazei Gö1 revealed the existence of two open reading frames encoding proton-translocating pyrophosphatases (PPases). These open reading frames are linked by a 750-bp intergenic region containing TC-rich stretches and are transcribed in opposite directions. The corresponding polypeptides are referred to as Mvp1 and Mvp2 and consist of 671 and 676 amino acids, respectively. Both enzymes represent extremely hydrophobic, integral membrane proteins with 15 predicted transmembrane segments and an overall amino acid sequence similarity of 50.1%. Multiple sequence alignments revealed that Mvp1 is closely related to eukaryotic PPases, whereas Mvp2 shows highest homologies to bacterial PPases. Northern blot experiments with RNA from methanol-grown cells harvested in the mid-log growth phase indicated that only Mvp2 was produced under these conditions. Analysis of washed membranes showed that Mvp2 had a specific activity of 0.34 U mg (protein)(-1). Proton translocation experiments with inverted membrane vesicles prepared from methanol-grown cells showed that hydrolysis of 1 mol of pyrophosphate was coupled to the translocation of about 1 mol of protons across the cytoplasmic membrane. Appropriate conditions for mvp1 expression could not be determined yet. The pyrophosphatases of M. mazei Gö1 represent the first examples of this enzyme class in methanogenic archaea and may be part of their energy-conserving system.
Highlights
Inorganic pyrophosphate (PPi) is formed in several enzymatic reactions of various metabolic pathways
Summary Analysis of genome sequence data from the methanogenic archaeon Methanosarcina mazei Gö1 revealed the existence of two open reading frames encoding protontranslocating pyrophosphatases (PPases)
Proton translocation experiments with inverted membrane vesicles prepared from methanol-grown cells showed that hydrolysis of 1 mol of pyrophosphate was coupled to the translocation of about 1 mol of protons across the cytoplasmic membrane
Summary
Inorganic pyrophosphate (PPi) is formed in several enzymatic reactions of various metabolic pathways (e.g., deoxyribonucleic acid and ribonucleic acid polymerization and amino acid and fatty acid activation). It is possible that pyrophosphatases utilize a portion of the PPi anhydride bond energy for the generation of a transmembrane proton gradient that could be used to drive endergonic reactions. The second class comprises tightly membrane-bound pyrophosphatases that were first isolated from vacuolar membranes of higher plants and algae (Maeshima 2000). These enzymes translocate protons across the vacuolar membrane, thereby maintaining the acidic vacuolar interior milieu. Proton-translocating pyrophosphatases have been found in the cytoplasmic membrane of several bacteria such as Rhodospirillum rubrum (Baltscheffsky et al 1998), Thermotoga maritima (Nelson et al 1999), Streptomyces coelicolor (Redenbach et al 1996), Synthrophus gentianae (Schöcke and Schink 1998) and in the hyperthermophilic archaeon Pyrobaculum aerophilum (Drozdowicz et al 1999, Drozdowicz and Rea 2001)
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