Member Institutions:

• Bowling Green State U.
• Cleveland State U.
• Kent State U.
• Medical College of Ohio
• Miami U.
• The Ohio State U.
• Ohio U.
• University of Cincinnati
• University of Toledo
• Wright State U.
• Youngstown State U.

Research Interests

Our lab seeks to understand the molecular and structural determinants of ribulose 1,5-bisphosphate carboxylase/oxygenase RubisCO specificity.  RubisCO is the enzyme of the Calvin-Benson-Bassham pathway that fixes CO₂ onto ribulose 1,5-bisphosphate (RuBP).  In a competing reaction, O₂ is fixed onto RuBP, initiating a photorespiratory pathway which ultimately results in carbon and energy loss for the organism.  CO₂ and O₂ fixation are related by a specificity factor (W) and the relative concentrations of the gaseous substrates in the vicinity of the enzyme:  v_c/v_o = W * [CO₂]/[O₂].  Understanding the evolutionary conservation of the seemingly wasteful photorespiratory pathway requires an understanding of the structural and functional determinants of RubisCO specificity.

We are using Ralstonia eutropha (formerly Alcaligenes eutrophus), an aerobic, facultatively chemoautotrophic bacterium, to study RubisCO.   Unlike photosynthetic organisms, who harness light energy to fix CO₂, R. eutropha gleans energy from either hydrogen or formate for autotrophic growth.  During organoautotrophic growth, formate is oxidized to CO₂ that is fixed via RubisCO.  By altering growth conditions, such as the concentration of oxygen or formate, growth of R. eutropha on formate may be used for selection of mutations altering RubisCO specificity.  Similar systems of random mutagenesis and biological selection have shown great promise relative to the traditional approach of site directed mutagenesis (Spreitzer et al., 1995; Spreitzer and Salvucci, 2002; Smith and Tabita, 2003), but they are extremely limited by the rate of throughput from the time of mutagenesis to the time of identification of interesting mutations.   The ease with which R. eutropha can be genetically manipulated, as well as its relatively fast growth yield, will facilitate a high-throughput selection system that will yield novel RubisCO mutants.   Ultimately, we hope this deeper understanding of specificity may be translated by plant physiologists into increased plant productivity.

Selected Publications

• Smith, S.A. and F. R. Tabita. 2004. Glycine 176 affects catalytic properties and stability of the Synechococcus sp.strain PCC6301 ribulose-1,5-bisphosphate carboxylase/oxygenase. J. Biol. Chem. 279:25632-7.
• Smith, S.A. and F. R. Tabita. 2003. Positive and negative selection of mutant forms of prokaryotic (cyanobacterial) ribulose-1,5-bisphosphate carboxylase/oxygenase. J. Mol. Biol. 331:557-69.
• Spreitzer, R. J. and M. E. Salvucci. 2002. RUBISCO: Structure, regulatory interactions, and possibilities for a better enzyme. Annu. Rev. Plant Physiol. Plant Mol. Biol. 53: 449-475.
• Spreitzer, R. J., G. Thow and G. Zhu. 1995. Pseudoreversion substitution at large-subunit residue 54 influences the CO2/O2 specificity of chloroplast ribulose-bisphosphate carboxylase/oxygenase. Plant Physiol. 109: 681-685.

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