Highly active rubiscos discovered by systematic interrogation of natural sequence diversity

Area of Science:

• Biochemistry
• Enzymology
• Bioinformatics

Background:

• Carbon dioxide (CO2) conversion to biomass is primarily mediated by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco).
• Plant Rubiscos exhibit suboptimal carboxylation efficiency, prompting extensive kinetic characterization, yet only a small fraction of natural diversity has been explored.

Purpose of the Study:

• To identify and characterize novel, fast-carboxylating Rubisco variants from diverse genomic and metagenomic data.
• To expand the known kinetic capabilities of Rubisco beyond the limited studies on plant enzymes.

Main Methods:

• Systematic mining of genomic and metagenomic data to identify Rubisco sequences.
• Clustering of approximately 33,000 unique Rubisco sequences into ~1,000 similarity groups.
• In vitro synthesis, purification, and biochemical testing of 143 representative Rubisco variants.

Main Results:

• Over 100 synthesized Rubisco variants demonstrated in vitro activity.
• The fastest identified variant exhibited a turnover number of 22 ± 1 s−1, sixfold higher than the median plant Rubisco.
• Fastest variants were homodimers with simpler folding and activation kinetics compared to plant and cyanobacterial Rubiscos.

Conclusions:

• Discovery of significantly faster-carboxylating Rubisco variants expands the known functional diversity of this crucial enzyme.
• The developed pipeline offers a scalable approach for exploring the kinetic potential of other enzymes within the biosphere.
• These findings have implications for understanding and potentially enhancing carbon fixation processes.