Evolutionarily divergent, Na+-regulated H+-transporting membrane-bound pyrophosphatases

Area of Science:

• Biochemistry
• Molecular Biology
• Membrane Transport

Background:

• Membrane-bound pyrophosphatases (mPPases) utilize pyrophosphate (PPi) hydrolysis to power ion transport across cellular membranes.
• H+-transporting PPases are classified into K+-independent and K+-dependent subfamilies based on phylogenetic analysis.

Purpose of the Study:

• To characterize a novel group of 46 bacterial and 1 archaeal proteins distantly related to known mPPases.
• To investigate the functional and regulatory properties of these newly identified mPPase candidates.

Main Methods:

• Bioinformatic analysis to identify distantly related mPPase homologs.
• Protein expression in Escherichia coli.
• Enzymatic assays using inverted membrane vesicles to measure PPi hydrolysis and ion transport.
• Kinetic analyses of enzyme activity under varying substrate and ion concentrations.

Main Results:

• A novel subfamily of H+-transporting mPPases was identified, sharing conserved residues with canonical mPPases but exhibiting distant evolutionary relationships.
• Expressed proteins catalyzed Mg2+-dependent PPi hydrolysis coupled to electrogenic H+ transport, but not Na+ transport.
• These novel H+-PPases are uniquely inhibited by Na+ and regulated by K+ in a PPi-dependent manner, suggesting alkali cations displace Mg2+ from the enzyme.

Conclusions:

• The identified proteins represent a new subfamily of H+-transporting membrane-bound pyrophosphatases.
• This subfamily exhibits unique regulatory mechanisms involving Na+ and K+ ions, potentially reflecting ancestral regulatory patterns from Na+-transporting mPPase precursors.