A novel prokaryotic phospholipase A2. Characterization, gene cloning, and solution structure

Area of Science:

• Biochemistry
• Microbiology
• Structural Biology

Background:

• Phospholipase A(2) (PLA(2)) enzymes were previously exclusively identified in eukaryotic organisms.
• The discovery of PLA(2) in prokaryotes expands the known biological sources of this enzyme class.

Purpose of the Study:

• To identify and characterize a novel phospholipase A(2) from a prokaryotic source.
• To elucidate the structural and biochemical properties of this first-ever prokaryotic PLA(2).

Main Methods:

• Isolation and characterization of secreted PLA(2) from Streptomyces violaceoruber.
• Gene cloning, sequencing, and overexpression in a Streptomyces host-vector system.
• Nuclear Magnetic Resonance (NMR) spectroscopy for solution structure determination.
• Calcium-binding experiments to determine dissociation constants.

Main Results:

• Identified and characterized the first prokaryotic secreted PLA(2) from Streptomyces violaceoruber.
• The prokaryotic PLA(2) possesses a unique structure with only four cysteines and five alpha-helices, differing significantly from eukaryotic PLA(2)s.
• Determined the solution structures of the enzyme, both with and without calcium ions, revealing distinct calcium-binding domain features.
• The prokaryotic PLA(2) exhibits a larger calcium dissociation constant compared to eukaryotic PLA(2)s.

Conclusions:

• This study reports the first identification of phospholipase A(2) in prokaryotes, specifically from Streptomyces violaceoruber.
• The prokaryotic PLA(2) displays a distinct structural fold and calcium-binding characteristics compared to known eukaryotic enzymes.
• The findings broaden the understanding of PLA(2) evolution and distribution across different domains of life.