A single amino acid substitution increases both carboxylation turnover number and CO2 affinity of form II Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key CO2-fixing enzyme in photosynthesis, is notorious for its low carboxylation activity. However, the difficulty in rationally engineering a fast Rubisco over the past decades brings a question whether a constraint exists in Rubisco's catalytic mechanism. In this study, we show that altering a single amino acid at position 398 in Form II Rubisco doubles its catalytic efficiency. The T398S and T398A mutations of the Form II Rubisco from the symbiont of Riftia pachyptila increases activity by 61 % and 74 %, respectively. The T398A mutant exhibits a turnover number (kcatC) of 35.84 s-1, twice that of the wild type. Structural simulation analysis indicates that the distance between the amino acid residues at position 398 and 395 influences weak hydrogen bond formation. Remarkably, these enhancements were achieved without compromising CO2 affinity (KMC), challenging the conventional trade-off paradigm. Our findings not only identify residue 398 as a critical determinant of Rubisco's performance but also highlight the untapped potential for engineering more efficient CO2-fixing enzymes.