Pinpointing Synechococcus Rubisco large subunit sections involved in heterologous holoenzyme formation in Escherichia coli

ABSTRACT

Aims@#Heterologous holoenzyme formation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has been a challenge due to a limited understanding of its biogenesis. Unlike bacterial Rubiscos, eukaryotic Rubiscos are incompatible with the Escherichia coli (E. coli) chaperone system to fold and assemble into the functional hexadecameric conformation (L8S8), which comprises eight large subunits (RbcL) and eight small subunits (RbcS). Our previous study reported three sections (residues 248-297, 348-397 and 398-447) within the RbcL of Synechococcus elongatus PCC6301, which may be important for the formation of L8S8 in E. coli. The present study further examined these three sections separately, dividing them into six sections of 25 residues (i.e., residues 248-272, 273-297, 348-372, 373-397, 398-422 and 423-447).@*Methodology and results@#Six chimeric Rubiscos with each section within the RbcL from Synechococcus replaced by their respective counterpart sequence from Chlamydomonas reinhardtii were constructed and checked for their effect on holoenzyme formation in E. coli. The present study shows that Section 1 (residues 248-272; section of Synechococcus RbcL replaced by corresponding Chlamydomonas sequence), Section 2 (residues 273-297), Section 3 (residues 348-372) and Section 6 (residues 423-447) chimeras failed to fold and assemble despite successful expression of both RbcL and RbcS. Only Section 4 (residues 373-397) and 5 (residues 398-422) chimeras could form L8S8 in E. coli.@*Conclusion, significance and impact of study@#GroEL chaperonin mediates the folding of bacterial RbcL in E. coli. Therefore, residues 248-297, 348-372 and 423-447 of Synechococcus RbcL may be important for interacting with the GroEL chaperonin for successful holoenzyme formation in E. coli.