Abstract
A previously described system for biological selection of randomly mutagenized ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) employing the phototrophic bacterium Rhodobacter capsulatus was used to select a catalytically altered form of a cyanobacterial (Synechococcus sp. strain PCC6301) enzyme. This mutant Rubisco, in which conserved glycine 176 was replaced with an aspartate residue, was not able to support CO(2)-dependent growth of the host strain. Site-directed mutant proteins were also constructed, e.g. asparagine and alanine residues replaced the native glycine with the result that these mutant proteins either greatly reduced the ability of R. capsulatus to support growth or had little effect, respectively. Growth phenotypes were consistent with the Rubisco activity levels associated with these proteins, and this was also borne out with purified recombinant proteins. Despite being catalytically challenged, the G176D and G176N mutant proteins were found to exhibit a more favorable interaction with CO(2) than the wild type protein but exhibited a reduced affinity for the substrate ribulose 1,5-bisphosphate. The G176A enzyme differed little from the wild type protein in these properties. None of the mutants had CO(2)/O(2) specificities that differed markedly from the wild type. Further studies taken from the known structure of the Synechococcus Rubisco indicated that substitutions at Gly-176 affected associations between large subunits. Supporting experimental data included an unusual protein concentration-dependent effect on in vitro activity, differences in thermal stability relative to the wild type protein, and aberrant migration on nondenaturing polyacrylamide gels. From these results, it is apparent that residues not directly located within the active site but near large subunit interfaces can affect key kinetic properties of Rubisco. These results suggest that further bioselection protocols (using these proteins as starting material) might yield novel mutant forms of Rubisco that relate to key functional properties.
Highlights
A system was described for directed evolution of prokaryotic Rubisco genes using the purple, nonsulfur, phototrophic bacterium Rhodobacter capsulatus as a selective host [3]
In this report we show that the kinetic properties (Kc and KRuBP) of the G176D and G176N mutant proteins were greatly altered
Residue glycine 176 (Gly-176) and Growth Phenotypes—In a previous report [3], a system was described whereby potentially interesting mutant forms of cyanobacterial Rubisco could be selected after random mutagenesis and complementation of a Rubiscodepleted strain of R. capsulatus
Summary
Plasmids, and Growth—Construction of the Rubisco deletion strain of R. capsulatus strain SBI-IIϪ was described previously [4]. Plasmid pRPS-MCS3 was constructed for this selection system It was derived from the broad host range plasmid pBBR1-MCS3 [13] and contained a Rubisco promoter from Rhodospirillum rubrum to express wild type and mutated rbcLS genes in R. capsulatus SBI-IIϪ. Mated cells were plated onto peptone yeast extract with rifampicin to select against Ep. coli and tetracycline to select against R. capsulatus that had not received the pRPS-MCS3 plasmid. These plates were incubated for 3 days at 30 °C. 1-liter cultures of Es. coli JM109 containing pUC19 subclones of the wild type and mutant enzymes were induced with 0.5 mM isopropyl-1-thio--Dgalactopyranoside overnight, and harvested cells were disrupted by sonication. Samples were assayed by the standard protocol with 1-min assays for the wild type, K11R, and G176A enzymes and 5-min assays for the
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