About this Journal Submit a Manuscript Table of Contents
Archaea
Volume 1 (2002), Issue 1, Pages 1-7
http://dx.doi.org/10.1155/2002/371325
Research Article

Identification and analysis of proton-translocating pyrophosphatases in the methanogenic archaeon Methanosarcina mazei

1Abteilung Allgemeine Mikrobiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität, Grisebachstr. 8, 37077 Göttingen, Germany
2Göttingen Genomics Laboratory, Institut für Mikrobiologie und Genetik, Georg-August-Universität, Grisebachstr. 8, 37077 Göttingen, Germany

Received 12 June 2001; Accepted 14 August 2001

Copyright © 2002 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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. Abbreviations: DCCD, N,N′-dicyclohexylcarbodiimide; PPase, inorganic pyrophosphatase; PPi, inorganic pyrophosphate; Δp, proton motive force.