Linked Rubisco subunits can assemble into functional oligomers without impeding catalytic performance.

Area of Science:

• Biochemistry
• Molecular Biology
• Plant Science

Background:

• Transgenic manipulation of the large subunit (L) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is feasible in plants.
• Engineering the small subunit (S) of Rubisco is challenging due to its multiple nuclear gene copies.
• Simultaneous engineering of both L and S subunits could offer a novel approach to Rubisco modification.

Purpose of the Study:

• To investigate the feasibility of simultaneously engineering Rubisco L and S subunits by creating fusion peptides.
• To assess the assembly, structure, and catalytic activity of these LS fusion proteins.
• To establish a framework for alternative strategies in modifying Rubisco subunits in plant plastids.

Main Methods:

• Fusion peptides of Synechococcus PCC6301 Rubisco L and S subunits were constructed using various linker sequences.
• Variant fusion peptides were expressed in Escherichia coli to analyze their assembly into functional Rubisco complexes.
• Catalytic properties (CO2/O2 specificity, carboxylation rate, Michaelis constants) of assembled complexes were measured.

Main Results:

• Synechococcal LS fusions assembled into catalytically functional octameric and hexadecameric structures in E. coli.
• Assembly efficiency of LS fusions was reduced compared to unlinked L and S subunits.
• Fusions with S linked to the N-terminus of L exhibited catalytic properties similar to wild-type Rubisco, while other fusion orientations showed compromised activity.

Conclusions:

• Tethering Rubisco L and S subunits is a viable strategy for creating functional, albeit less efficiently assembled, Rubisco complexes.
• The orientation of the fusion significantly impacts the catalytic performance of the engineered Rubisco.
• This work presents a promising framework for the simultaneous engineering of modified or foreign Rubisco subunits in higher plant plastids.