Poster: Photosynthesis

Abs # 326: Identification of a novel intracellular b - carbonic anhydrase in Chlamydomonas reinhardtii which is distinct from the mitochondrial forms of the enzyme

Presenter:	Mitra, Mautusi , mmitra@lsu.edu
Authors	Mitra, Mautusi  (A)   Ynalvez, Ruby A. (A)   Lato, Scott M. (A)   Moroney, James V. (A)
Affiliations:	(A): Department of Biological Sciences, Louisiana State University

Carbonic anhydrases (CA) are zinc containing metalloenzymes that catalyze the reversible hydration of CO₂. The three evolutionarily unrelated families of carbonic anhydrases are designated a-, b- and g- CA. Aquatic photosynthetic organisms have evolved different forms of CO₂ concentrating mechanisms (CCMs) to aid Rubisco in capturing CO₂ from the surrounding environment. One aspect of all CCMs is the critical roles played by various specially localized extracellular and intracellular CAs. Five carbonic anhydrases have previously been identified in Chlamydomonas reinhardtii, a green alga with a well studied CCM. We have identified a sixth gene encoding a b- type CA. This new b-CA, designated BETA CA3, is distinct from the two mitochondrial b-CAs and has a putative chloroplast leader sequence. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 264 amino acids. We have fused the BETA CA3 cDNA to the coding sequence of maltose binding protein (MBP) in a pMal expression vector. The protein was overexpressed as a recombinant fusion protein and purified by amylose affinity column chromatography. The purified recombinant fusion protein was cleaved with protease Factor Xa to separate BETA CA3 from the MBP. The BETA CA3 protein was then used to raise an antibody and to study some of its biochemical properties. We have also shown that the recombinant fusion protein is active. Supported by NSF grants IBN-9904425 and IBN-0212093