Synthetic DNA system for structure-function studies of the high affinity CO₂ uptake NDH-1₃ protein complex in...

The CO₂-concentrating mechanism (CCM) in cyanobacteria supports high rates of photosynthesis by greatly increasing the concentration of CO₂ around the major carbon fixing enzyme, Rubisco. However, the CCM remains poorly understood, especially in regards to the enigmatic CO₂-hydration enzymes which couple photosynthetically generated redox energy to the hydration of CO₂ to bicarbonate. This CO₂-hydration reaction is catalysed by specialized forms of NDH-1 thylakoid membrane complexes that contain phylogenetically unique extrinsic proteins that appear to couple CO₂ hydration to NDH-1 proton pumping. The development of the first molecular genetic system to probe structure-function relationships of this important enzyme system is described. A CO₂-hydration deficient strain was constructed as a recipient for DNA constructs containing different forms of the CO₂-hydration system. This was tested by introducing a construct to an ectopic location that gives constitutive expression, rather than native inducible expression, of the ndhF3-ndhD3-cupA-cupS, (cupA operon) encoding high affinity CO₂-hydration complex, NDH-1₃. Uptake assays show the restoration of high affinity for CO₂ uptake, but demonstrate that the CupA complex can drive only modest uptake fluxes, underlining the importance of its tandem operation with the CupB-containing complex NDH-1₄, the complementary high flux, low affinity CO₂ hydration system. Experiments with the carbonic anhydrase inhibitor, ethoxyzolamide, indicate that the NDH-1₃ complex is strongly inhibited, yet the remaining NDH-1₄ activity in the wild-type is less so, suggesting structural differences between the low affinity and high affinity CO₂-hydration systems. This new construct will be an important tool to study and better understand cyanobacterial CO₂ uptake systems.