Occurrence of diphthamide in archaebacteria.

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Diphtheria toxin fragment A (DTA) ribosylates elongation factors, inhibiting protein synthesis.
• Eukaryotic elongation factor 2 (EF-2) is the known substrate for DTA.
• Archaebacteria possess unique biological characteristics, differing from eukaryotes and eubacteria.

Purpose of the Study:

• To investigate the interaction of DTA with protein synthesis factors from archaebacteria.
• To determine if archaebacterial factors contain diphthamide, a modification found in eukaryotic EF-2.
• To compare the DTA recognition site and diphthamide presence between archaebacterial and eukaryotic systems.

Main Methods:

• Cell-free extracts from Thermoplasma acidophilum and Halobacterium halobium were prepared.
• ADP-ribosylation assays were performed using DTA and archaebacterial extracts.
• Acid hydrolysis of proteins followed by analysis for diphthine was conducted.
• Comparative analysis with eukaryotic (Saccharomyces cerevisiae, HeLa) and eubacterial (Escherichia coli) proteins.

Main Results:

• Archaebacterial extracts contained a protein factor that served as a substrate for DTA-mediated ADP-ribosylation.
• The reaction rate with archaebacterial factors was approximately 1,000 times slower than with eukaryotic EF-2.
• Diphthine was detected in Halobacterium halobium protein hydrolysates, similar to eukaryotic cells.
• Diphthine was absent in Escherichia coli protein hydrolysates.

Conclusions:

• Archaebacterial protein synthesis factors are recognized by DTA, albeit with significantly reduced efficiency compared to eukaryotes.
• The presence of diphthamide in archaebacteria, but not in eubacteria, suggests an evolutionary link to eukaryotes.
• While sharing the diphthamide modification, archaebacterial and eukaryotic elongation factors exhibit functional differences in DTA interaction.