Characterization of the carbonic anhydrase isozymes of Zea mays

In maize, CA catalyzes the first reaction in the C4 photosynthetic pathway, hydrating carbon dioxide that has diffused into the mesophyll cell cytoplasm to bicarbonate, providing an inorganic carbon source for the C4 pathway. The beta-CA isozymes from maize, as well as other agronomically important C4 crops such as sorghum and sugarcane, differ significantly from other reported forms of the enzyme and have remained relatively uncharacterized. The mRNA transcripts encoding the CA isozymes contain repeating sequences of approximately 600 bp that encode multiple protein domains (Repeat A, Repeat B and Repeat C). In maize, three cDNA sequences had been determined and designated CA1, CA2 and CA3. There are at least three genes in the maize genome, and one of these encodes two identical protein domains, with distinct groups of exons corresponding to the repeating regions of the transcript. The first exon of the CA2 gene encodes a putative chloroplast transit peptide, indicating an additional non-photosynthetic role for CA in maize, such as in lipid biosynthesis pathways and/or replenishing the Krebs cycle intermediates together with PEP carboxylase. This is supported by the identification of CA transcripts in root tissue and analysis of the gene sequence, which identified promoter elements that direct constitutive expression. The expression of a single repeat region of the transcript produced active enzyme, able to catalyze the reversible hydration of carbon dioxide to bicarbonate producing hydrogen ions. The carbon dioxide hydration activity of Repeat B was relatively high compared to the activity of either Repeat A or C. Repeat B was also found to be a dimer and is composed primarily of alpha-helices, in agreement with that observed for other plant CAs. The active site of the individual protein domains, Repeat A, Repeat B and Repeat C was identified and found to contain the conserved amino acids proposed to coordinate the catalytic zinc ion and act as a proton acceptor during regeneration of the active enzyme complex