Screening of hyperthermostable proteins from hyperthermophilic archaeon Palaeococcus pacificus DY20341T.

Abstract

Palaeococcus pacificus DY20341^T, a hyperthermophilic archaeon from deep sea hydrothermal sediments, grows at temperature range from 50 to 90 ℃ (Topt = 80℃). To explore the molecular basis of its thermal adaptation, we identified six representative hyperthermostable proteins from its cytosolic fraction that remained soluble after a stringent in vitro heat treatment at 100 °C. MALDI-TOF mass spectrometry identified these as an inorganic pyrophosphatase (PPase), a 5'-methylthioadenosine phosphorylase (MTAP), and four putative transporter components (one TRAP and three ABC transporter solute-binding proteins). RT-qPCR analysis revealed that the transcription of all six corresponding genes was significantly upregulated when the growth temperature was elevated from 80 °C to 90 °C, suggesting a potential role in the cellular response to heat stress. Biochemical characterization of the recombinant PPase demonstrated remarkable thermostability and an optimal temperature of 90 °C. Kinetic analysis revealed high catalytic efficiency, with a maximal specific activity of 590 ± 30 U/mg and a strong substrate affinity ([Formula: see text] = 6.24 µM). Phylogenetic analysis of the identified thermostable MTAP (PAP_00250) revealed its evolutionary origin from hyperthermophilic archaea, distinguishing it clearly from a second MTAP homolog (PAP_07050) in the genome, which aligns with mesophilic bacteria. The structural stability of the identified ABC and TRAP transporter components hypothesizes that efficient nutrient scavenging remains critical at extreme temperatures. Together, these findings provide valuable insights into the stable molecular machinery and potential physiological strategies enabling P. pacificus to thrive in hydrothermal environments.