title: Linked Rubisco subunits can assemble into functional oligomers without impeding catalytic performance.
abstract: Although transgenic manipulation in higher plants of the catalytic large subunit (L) of the photosynthetic CO2-fixing enzyme ribulose 1,5-bisphospahte carboxylase/oxygenase (Rubisco) is now possible, the manipulation of its cognate small subunit (S) is frustrated by the nuclear location of its multiple gene copies. To examine whether L and S can be engineered simultaneously by fusing them together, the subunits from Synechococcus PCC6301 Rubisco were tethered together by different linker sequences, producing variant fusion peptides. In Escherichia coli the variant PCC6301 LS fusions assembled into catalytically functional octameric ([LS]8) and hexadecameric ([[LS]8]2) quaternary structures that excluded the integration of co-expressed unfused S. Assembly of the LS fusions into Rubisco complexes was impaired 50-90% relative to the assembly of unlinked L and S into L8S8 enzyme. Assembly in E. coli was not emulated using tobacco SL fusions that accumulated entirely as insoluble protein. Catalytic measurements showed the CO2/O2 specificity, carboxylation rate, and Michaelis constants for CO2 and ribulose 1,5-bisphosphate for the cyanobacterial Rubisco complexes comprising fusions where the S was linked to the N terminus of L closely matched those of the wild-type L8S8 enzyme. In contrast, the substrate affinities and carboxylation rate of the Rubisco complexes comprising fusions where L was fused to the N terminus of S or a six-histidine tag was appended to the C terminus of L were compromised. Overall this work provides a framework for implementing an alternative strategy for exploring simultaneous engineering of modified, or foreign, Rubisco L and S subunits in higher plant plastids.
authors.affiliation: Molecular Plant Physiology, Research School of Biological Sciences, Australian National University, P O Box 475, Canberra, Australian Capital Territory 2601, Australia. spencer.whitney@anu.edu.au
authors.forename: Spencer M
authors.initials: SM
authors.lastname: Whitney
authors.forename: Robert E
authors.initials: RE
authors.lastname: Sharwood
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics; D015202:Protein Engineering; D017510:Protein Folding; D021122:Protein Subunits / Q000378:metabolism* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D046940:Synechococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D010455:Peptides; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012273:Ribulose-Bisphosphate Carboxylase