Part:BBa_K5478005

This composite part expresses for Ribulose-1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco) enzyme's co-evolved chaperone, RuBisCO Activase (RCA) found in Nicotiana Tabacum.

RuBisCO is the most abundant enzyme in the world as it is the key enzyme in photosynthesis, catalyzing the carbon dioxide fixation in the Calvin Cycle. It consists of a co-evolved chaperon known as RuBisCO Activase (RCA) which is critical for its catalytic abilities.

RCA works by removing the intrinsic sugar phosphate inhibitor, RuBP, from RuBisCO's active sites, allowing RuBisCO to maintain in its catalytically active form through ATPase activity. The removal of the inhibitor allows RuBisCO to combine RuBP with carbon dioxide, so it can split into two 3-phosphoglycerate (3PGA) molecules. The ATPase activity of RCA is regulated by “ADP/ATP ratio in the stroma because ADP inhibits the ATP hydrolysis reaction of the activase” (Zhang & Portis, 1999).

What is most intriguing is that most organisms which consist of a RCA, have two isoforms which are said to have “[arose] from alternative splicing [of the gene transcript] that differ only at the carboxyl terminus” (Zhang & Portis, 1999). RCA1 Nicotiana Tabacum is the smaller of the two isoforms of RCA found in Nicotiana Tabacum.

That being said, one of RCA’s limitations is its thermosensitivity. In fact, at temperatures just above the thermal optimum of net carbon dioxide assimilation rate (typically 30°C to 40°C), RCA complexes dissociate and begin denaturing. Though this phase is reversible upon cooling, in temperatures above about 42°C, RCA denatures while RuBisCO doesn’t. This results in a bottleneck, where RCA’s thermosensitivity limits Rubisco’s activity (Salvucci, 2001).

In this specific component, the chloroplast localization tag from the RCA was removed. It also consists of a T7 promoter, 5’ UTR, and T7 terminator with Type IIS Restriction Sites coding for BsaI at either ends. NOTE: THIS IS NOT THE CODON OPTIMIZED VERSION.

Zhang, N., & Portis, A. R. (1999, August 3). Mechanism of light regulation of Rubisco: A specific role for the larger Rubisco activase isoform involving reductive activation by thioredoxin-f. PNAS. Retrieved June 8, 2024, from https://www.pnas.org/doi/10.1073/pnas.96.16.9438#:~:text=Rubisco%20activase%20is%20a%20nuclear,oxygenase%20(Rubisco)%20in%20vivo Salvucci, M. E., Osteryoung, K. W., Crafts-Brandner, S. J., & Vierling, E. (2001). Exceptional sensitivity of Rubisco activase to thermal denaturation in vitro and in vivo. Plant physiology, 127(3), 1053–1064.