Effects of mutations at residue 309 of the large subunit of ribulosebisphosphate carboxylase from Synechococcus PCC 6301

Abstract

Previous studies [G. S. Hudson et al. (1989) J. Biol. Chem. 265, 808–814] showed that the faster turnover rates and lower affinities for CO 2 of ribulosebisphosphate carboxylase/oxygenases from C 4 plants, compared to C 3 and C 3 C 4 plants, were specified by the chloroplast-encoded large subunits. In pairs of closely related C 3 and C 4 species from three genera, these kinetic changes were accompanied by only three to six amino acid residue substitutions, depending on the genus. None of these substitutions occurred near the active site and only one, 309Met (C 3) to Ile (C 4), was common to all three genera. Unlike the plant carboxylases, the highly homologous enzyme from the cyanobacterium Synechococcus PCC 6301 folds and assembles properly when its rbcL and rbcS genes are coexpressed in Escherichia coli. Furthermore, the cyanobacterial enzyme has Ile at position 309 of the large subunit, a high turnover number, and a poor affinity for CO 2. 309Ile was replaced with Met and several other residues by site-directed mutagenesis of the cyanobacterial rbcL. Met and Leu were tolerated at this position with no alteration in the kinetic or structural properties of the assembled holoenzyme. However, substitution with Val, Gly, Trp, or Arg prevented the assembly of the subunits. The indifference to Met or Ile at this position, as well as the tolerance for Leu which is not observed with any natural ribulosebisphosphate carboxylase, leads to the conclusion that either the 309Met/Ile substitution has no effect on the kinetic properties of the plant enzyme, despite the correlation apparent in previous studies, or the cyanobacterial enzyme is sufficiently different from the plant enzyme in other respects that the influence of residue 309 is masked.