H + ‐proton‐pumping inorganic pyrophosphatase: a tightly membrane‐bound family

mentioning

confidence: 99%

Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer

Huang

Liu

Chen

et al. 2010

“…H ϩ -translocating inorganic pyrophosphatases (H ϩ -PPases) 1 consist of a single polypeptide. They are distinct from P-, F-, and V-type ATPases and are considered to be a fourth type of H ϩ pump (1,2).…”

mentioning

confidence: 99%

Membrane Topology of the H+-pyrophosphatase of Streptomyces coelicolor Determined by Cysteine-scanning Mutagenesis

Mimura

Nakanishi

Hirono

et al. 2004

The H ؉ -translocating pyrophosphatase (H ؉ -PPase) is a proton pump that is found in a wide variety of organisms. It consists of a single polypeptide chain that is thought to possess between 14 and 17 transmembrane domains. To determine the topological arrangement of its conserved motifs and transmembrane domains, we carried out a cysteine-scanning analysis by determining the membrane topology of cysteine substitution mutants of Streptomyces coelicolor H ؉ -PPase expressed in Escherichia coli using chemical reagents. First, we prepared a synthetic DNA that encoded the enzyme and constructed a functional cysteine-less mutant by substituting the four cysteine residues. We then introduced cysteine residues individually into 42 sites in its hydrophilic regions and N-and C-terminal segments. Thirtysix of the mutant enzymes retained both pyrophosphatase and H ؉ -translocating activities. Analysis of 29 of these mutant forms using membrane-permeable and -impermeable sulfhydryl reagents revealed that S. coelicolor H ؉ -PPase contains 17 transmembrane domains and that several conserved segments, such as the substrate-binding domains, are exposed to the cytoplasm. Four essential serine residues that were located on the cytoplasmic side were also identified. A marked characteristic of the S. coelicolor enzyme is a long additional sequence that includes a transmembrane domain at the C terminus. We propose that the basic structure of H ؉ -PPases has 16 transmembrane domains with several large cytoplasmic loops containing functional motifs.

“…is an integral membrane protein that utilizes the energy released upon hydrolysis of pyrophosphate (PP i ) to transport protons across the membrane against the electrochemical potential gradient (1)(2)(3). H ϩ -PPases represent a distinct class of ion translocases with no sequence similarity to ubiquitous ATP-energized pumps such as F-, V-, and P-type ATPases or ABC transporters (4).…”

mentioning

confidence: 99%

A Lysine Substitute for K+

Belogurov¹,

Lahti²

2002

The H+proton-translocating inorganic pyrophosphatase (H+-PPase) family is composed of two phylogenetically distinct types of enzymes: K+-dependent and K+-independent. However, to date, the sequence criteria governing this dichotomy have remained unknown. In this study, we describe the heterologous expression and functional characterization of H+-PPase from the thermophilic bacteriumCarboxydothermus hydrogenoformans. Both PPi-hydrolyzing and PPi-energized H+translocation activities of the recombinant enzyme inEscherichia coliinner membrane vesicles are strictly K+-dependent. Here we deduce the K+requirement of all available H+-PPase sequences based on the K+dependence ofC. hydrogenoformansH+-PPase in conjunction with phylogenetic analyses. Our data reveal that K+-independent H+-PPases possess conserved Lys and Thr that are absent in K+-dependent H+-PPases. We further demonstrate that a A460K substitution inC. hydrogenoformansH+-PPase is sufficient to confer K+independence to both PPihydrolysis and PPi-energized H+translocation. In contrast, a A463T mutation does not affect the K+dependence of H+-PPase.