RUI: Carbonic Anhydrases Involved in Algal Inorganic Carbon Acquisition

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The enzyme carbonic anhydrase catalyzes the interconversion of the two forms of inorganic carbon, carbon dioxide and bicarbonate ion, that predominate in aqueous media at physiological pH values. In the alga Chlamydomonas reinhardtii, one or more forms of carbonic anhydrase are components of a system for inorganic carbon acquisition and concentration. This system enables this and other algae to efficiently utilize low levels of carbon dioxide that could otherwise limit their growth. The role of a periplasmic carbonic anhydrase in inorganic carbon acquisition is well established, and there is substantial evidence that intracellular carbonic anhydrase activity is also essential for efficient inorganic carbon u tilization. However, the proteins responsible for this intracellular activity have not been specifically identified. We propose to utilize a novel carbonic anhydrase specific active site directed radiolabeled photoaffinity reagent as a tool both in the identification and isolation of physiologically important intracellular forms of carbonic anhydrase that are present in levels too low to be readily identified and quantified by other methods, and in studies leading to the identification of genes encoding intracellular carbonic anhydrase. Studies to apply this photoaffinity reagent to the identification and localization of carbonic anhydrases in cyanobacterial systems will also be initiated. In addition, we propose to continue studies to investigate the relationship between the subunit structure and disulfide bond reduction status on both the catalytic and inhibitor binding properties of the predominant extracellular form of carbonic anhydrase from Chlamydomonas reinhardtii. The overall objective of these studies is to enhance our understanding of the role of carbonic anhydrases as important components of the systems by which algal cells are able to efficiently concentrate inorganic carbon to provide carbon dioxide for photosynthesis. %%% The unicellular green alga Chlamydomonas reinhtardii and a variety of other aquatic photosynthetic organisms have developed systems that allow them to acquire atmospheric carbon dioxide much more effectively than most terrestrial plants. This is important because photosynthetic organisms utilize carbon dioxide for the light dependent synthesis of carbohydrates, which in turn leads to plant growth and biomass production. Furthermore, many plants can be growth limited because of the low levels of carbon dioxide that are present in the atmosphere. An understanding of how the highly efficient carbon dioxide acquisition systems work in organisms like Chlamydomonas reinhardtii, may be of value in learning how to improve carbon dioxide utilization in higher plants. Alt hough all of the details of the way in which this system functions in Chlamydomonas reinhardtii are not yet understood, it is apparent that a particular enzyme, carbonic anhydrase, is an important component of this process. Carbonic anhydrase accelerates the interconversion of carbon dioxide and bicarbonate, and this interconversion is important to facilitate the transfer of these species of inorganic carbon among cellular compartments, and plays a role in making carbon dioxide available to the chloroplast, the intracellular site of conversion of carbon dioxide to carbohydrates. In Chlamydomonas reinhardtii, there is considerable evidence that several different forms of this enzyme are important for the process of carbon dioxide utilization, and that these forms are found in different parts of the algal cell. It is a primary goal of this work to isolate the form of the enzyme that is present in the chloroplast. This in turn may lead to the identification of the gene that encodes this protein, and to an understanding of how the gene is turned on and off in response to environmental stimuli. Other parts of the work may lead to the identification of other forms of carbonic anhydrase that are present in other compartments of the algal cell. We believe that this information will significantly enhance our understanding of the efficient mechanism for carbon dioxide acquisition in Chlamydomonas reinhardtii.