Identifier

etd-1111103-120146

Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Carbonic anhydrase (CA) is a zinc containing metalloenzyme that catalyzes the reversible interconversion of CO2 and HCO3-. There are three evolutionarily unrelated CA families designated alpha, beta and gamma CA. Vertebrates have members of the alpha CA family, while higher plants, algae and cyanobacteria have members belonging to all three CA families. In the green alga, Chlamydomonas reinhardtii, five CAs have previously been identified including three alpha CAs and two beta CAs. This dissertation describes the identification and characterization of new CA genes from C. reinhardtii. Four new CA or CA like genes have been discovered including two beta CAs and two gamma CAs. Three CAs were investigated further including the alpha CA Cah3, one of the new beta CAs, Cah6; and a new gamma CA designated Gclp1 for gamma CA like protein. Cah3 is an alpha CA located in the thylakoid. Past studies with two Cah3 mutants, ca-1 and cia3 have shown that Cah3 plays an important role in the CO2 concentrating mechanism. In this work, the mature Cah3 protein was overexpressed as a fusion protein in E. coli and found to have significant CA activity. This is the first report of detection of CA activity in the Cah3 protein and its partial biochemical characterization. A novel beta CA (Cah6) and a putative gamma CA (Gclp1) gene were identified in C. reinhardtii. Gclp1 is one of two putative gamma CAs found in C. reinhardtii. Both the Cah6 and Gclp1 open reading frames (ORFs) were cloned in the overexpression vector pMal-c2x and expressed as recombinant fusion proteins. The purified Cah6 had significant in vitro CA activity but Gclp1 did not. Gclp1 was designated as a gamma CA like protein because it lacked detectable CA activity. Cah6 has a leader sequence consistent with a chloroplast localization. Although Cah6 is constitutively expressed under low and high CO2 conditions, it is slightly upregulated under low CO2 conditions. Immunolocalization studies confirmed that the Cah6 is localized to the chloroplast stroma particularly in the starch sheath around the pyrenoid. A possible role of Cah6 in the CO2 concentrating mechanism and photosynthesis is discussed.

Date

2003

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

James V. Moroney

DOI

10.31390/gradschool_dissertations.1701

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