Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity.
Marta GrzechowiakMilosz RuszkowskiJoanna SliwiakKamil SzpotkowskiMichal SikorskiMariusz JaskolskiPublished in: The Biochemical journal (2019)
Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31-Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.
Keyphrases
- induced apoptosis
- arabidopsis thaliana
- escherichia coli
- dna binding
- oxidative stress
- cell cycle arrest
- amino acid
- cell wall
- water soluble
- endoplasmic reticulum stress
- small molecule
- magnetic resonance
- perovskite solar cells
- cell death
- single cell
- cell free
- cancer therapy
- pseudomonas aeruginosa
- high resolution
- transcription factor
- magnetic resonance imaging
- staphylococcus aureus
- ms ms
- cell proliferation
- single molecule
- tandem mass spectrometry
- risk assessment
- saccharomyces cerevisiae
- high speed
- sewage sludge
- high performance liquid chromatography