Dimerization determines substrate specificity of a bacterial prenyltransferase.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Heptaprenylglyceryl phosphate synthase (PcrB) is crucial for isoprenoid biosynthesis in Bacillus subtilis.
• Oligomerization is often essential for protein function, but the specific role of PcrB dimerization was unclear.

Purpose of the Study:

• To identify the native dimer interface of PcrB.
• To investigate the impact of oligomerization on PcrB stability and catalytic activity.
• To understand how dimerization influences substrate specificity.

Main Methods:

• Computational prediction of potential dimer interfaces.
• In silico and experimental validation using amino acid substitutions and unnatural amino acid cross-linking.
• Thermal unfolding assays to assess protein stability.
• Enzyme activity assays to determine substrate specificity.

Main Results:

• The native dimer interface of PcrB was identified and confirmed to be conserved with homologous archaeal enzymes.
• Monomeric PcrB showed only a minor decrease in stability compared to the wild-type dimer.
• Monomerization significantly restricted the substrate chain length accepted by PcrB, limiting it to three isoprene units.

Conclusions:

• PcrB dimerization is critical for its native substrate specificity, particularly for accepting longer polyprenyl pyrophosphates.
• The identified dimer interface provides insights into the structural basis of PcrB function.
• Dimerization, rather than stability, appears to be the primary driver for PcrB's broad substrate specificity.