Structural studies of the prokaryotic carbon-concentrating mechanism

• Applicant: Dr. Jan Michael Schuller
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Biochemistry
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 437114812

The bioavailability of CO2 is a major growth-limiting factor for cyanobacteria and chemoautotrophic proteobacteria. Consequently, these prokaryotes have evolved a highly effective carbon-concentrating mechanism (CCM) that uses active and facilitated systems to accumulate high cellular levels of bicarbonate (HCO3-) at far from equilibrium concentrations. Carbonic anhydrases in carboxysomes then convert the bicarbonate to CO2 for Rubisco, the major carbon-fixing enzyme. Under alkaline growth conditions, extracellular HCO3- is taken up by active and secondary transport. SbtA, an inducible Na+ gradient-driven Na+/HCO3- symporter with high affinity to HCO3-, plays a major role in this process. SbtA is encoded on the same operon as SbtB, which has been shown to bind to SbtA and is proposed to regulate its activity. Under neutral and acidic conditions, the majority of the inorganic carbon is present as CO2, which diffuses into the cell and is subsequently captured by a unique uptake system that uses specialized forms of the photosynthetic complex I (NDH-1) the NDH-1MS and NDH-1MS’ complexes, to drive the conversion of CO2 to HCO3- against the equilibrium. The Cup proteins are the distinguishing feature of the carbon-capturing NDH-1 complexes and, although they have little sequence homology to other known carbonic anhydrases, they are responsible for the carbonic anhydrase activity of the complexes. The only other molecular machine that shows a recognizable functional relationship with the Cup proteins is the two-protein dissolved inorganic carbon-concentrating transporter (DIC-CT) system, which was discovered in T. crunogena but is widespread in archea and multiple phyla of bacteria, including human pathogens. The activity of the DIC-CT system requires an intact membrane potential; thus, the system likely represents a simplified version of the carbon capturing NDH-1 complexes. In this proposal, I will combine structural studies by cryo-electron microscopy single-particle analysis with reconstituted in vitro functional assays to obtain an integrated view of the prokaryotic CCM that spans scales from the atomic to the functional level. I will answer several important biological questions: 1) How is the SbtA transporter regulated by SbtB? 2) How can the NDH-1 complexes capture CO2 using redox-driven proton-pumping machinery, thereby revolutionarizing our understanding of complex I? 3) How does the two-protein DIC-CT transporter use a cation gradient to perform the same biological function as the multi-protein NDH-1 complexes? The structural and functional insights obtained from this study will transform biotechnological and synthetic biology applications, serving as a blueprint to generate improved industrial microbes for the production of chemical compounds, to create solar fuels, and to counteract global warming by sequestering CO2.

• DFG Programme: Independent Junior Research Groups