A Rubisco Mutant That Confers Growth Under a Normally "inhibitory" Oxygen Concentration

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) is a globally significant biocatalyst that facilitates the removal and sequestration of CO2 from the biosphere. Rubisco-catalyzed CO2 reduction thus provides virtually all of the organic carbon utilized by living organisms. Despite catalyzing the rate-limiting step of photosynthetic and chemoautotrophic CO2 assimilation, Rubisco is markedly inefficient as the competition between O2 and CO2 for the same substrate limits the ability of aerobic organisms to obtain maximum amounts of organic carbon for CO2-dependent growth. Random and site-directed mutagenesis procedures were coupled with genetic selection to identify an "oxygen-insensitive" mutant cyanobacterial (Synechococcus sp. strain PCC 6301) Rubisco that allowed for CO2-dependent growth of a host bacterium at an oxygen concentration that inhibited growth of the host containing wild-type Synechococcus Rubisco. The mutant substitution, A375V, was identified as an intragenic suppressor of D103V, a negative mutant enzyme incapable of supporting autotrophic growth. Ala-375 (Ala-378 of spinach Rubisco) is a conserved residue in all form I (plant-like) Rubiscos. Structure-function analyses indicate that the A375V substitution decreased the enzyme's oxygen sensitivity (and not CO2/O2 specificity), possibly by rearranging a network of interactions in a fairly conserved hydrophobic pocket near the active site. These studies point to the potential of engineering plants and other significant aerobic organisms to fix CO2 unfettered by the presence of O2.