Cloning And Characterization of an Enzyme Integral To Photosynthetic Assimilation of Atmospheric Carbon Dioxide

  The seemingly trite bumper sticker, "have you hugged a plant today?", actually reflects the profound truth that the entire animal kingdom requires plants for survival. We depend on plants for oxygen that we breathe, food that we eat, shelter that protects us from the elements, and medicinals that ward off disease. Fossilized plants fuel our automobiles, generate electricity, and provide innumerable consumer products and industrial materials. All of these benefits are consequences of the photosynthetic machinery by which plants capture and utilize the energy from sunlight to produce carbohydrates from atmospheric carbon dioxide (CO2). Photosynthetic conversion of atmospheric CO2 to carbohydrates, which is the only biospheric avenue for sequestration of this predominant greenhouse gas, is dependent on the concerted action of multiple enzymes that comprise the Calvin cycle. As inefficiency of this biosynthetic pathway severely curtails potential plant growth and yield, application of genetic engineering toward improved efficiency offers the prospect of enhanced biomass for energy, food, and global carbon management. Reaching such an ambitious, long-term goal is predicated on comprehensive understanding of mechanism and interplay of the requisite enzymes. Although some of the Calvin cycle enzymes are well-characterized, others have been glaringly neglected. For example, ribulose-5-phosphate epimerase, which is essential for the regeneration of the substrate for CO2 fixation and also provides for critical linkage between distinct metabolic pathways, has never even been isolated from plants due to its extreme instability and low natural abundance. Frank Larimer, Fred Hartman, and their coworkers of ORNL's Life Sciences Division have overcome these impediments by cloning the gene that encodes the spinach epimerase, expressing the gene in Escherichia coli, identifying conditions for stabilizing the epimerase, and isolating the overproduced enzyme. Structural, catalytic, and stability parameters of the purified, biologically-active epimerase have been characterized, thereby paving the way for future mechanistic studies. A manuscript describing these recent accomplishments has been submitted to a peer-reviewed journal.

Contact: Fred C. Hartman
Phone: (423) 574-0959
E-mail: ffh@ornl.gov