Synthetic DNA system for structure-function studies of the high affinity CO uptake NDH-1 protein complex in cyanobacteria.

The CO-concentrating mechanism (CCM) in cyanobacteria supports high rates of photosynthesis by greatly increasing the concentration of CO around the major carbon fixing enzyme, Rubisco. However, the CCM remains poorly understood, especially in regards to the enigmatic CO-hydration enzymes which couple photosynthetically generated redox energy to the hydration of CO to bicarbonate. This CO-hydration reaction is catalysed by specialized forms of NDH-1 thylakoid membrane complexes that contain phylogenetically unique extrinsic proteins that appear to couple CO 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 CO-hydration deficient strain was constructed as a recipient for DNA constructs containing different forms of the CO-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 CO-hydration complex, NDH-1. Uptake assays show the restoration of high affinity for CO 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-1, the complementary high flux, low affinity CO hydration system. Experiments with the carbonic anhydrase inhibitor, ethoxyzolamide, indicate that the NDH-1 complex is strongly inhibited, yet the remaining NDH-1 activity in the wild-type is less so, suggesting structural differences between the low affinity and high affinity CO-hydration systems. This new construct will be an important tool to study and better understand cyanobacterial CO uptake systems.