Isolation and characterization of a γ-carbonic anhydrase localized in the mitochondria of Saccharina japonica

Abstract Saccharina japonica is an ecologically and economically important seaweed that is dominant in the rocky shores of cold-temperate regions, forms the major component of productive beds, and affects marine environments. S. japonica exhibits a high photosynthetic efficiency in natural seawater with low dissolved CO2 concentration, thus suggesting the presence of its carbon-concentrating mechanism (CCM). However, the genes, proteins, and pathways involved in the CCM of S. japonica have not been fully identified and characterized. Carbonic anhydrase (CA) is a crucial component of CCM in macroalgae. In this study, the cloning, characterization, and subcellular localization of a specific CA were described. Multisequence alignment and phylogenetic analysis indicated that this CA belonged to the gamma (Sjγ-CA) class. This enzyme has a full-length cDAN of 1370 bp, encodes a protein with 246 amino acids (aa; ca. 25.7 kDa), and contains the mitochondrial transit peptide of 16 aa and LbH_gama_CA_like domain of 159 aa that defined the γ-CA region. The Sjγ-CA was successfully expressed in E. coli BL21 and purified as an active recombinant CA. Immunogold electron microscopy and fluorescence localization illustrated that this enzyme is localized in the mitochondria, and its transcription level is up-regulated by low CO2 concentration. These findings showed that Sjγ-CA is a possible component of the CCM in S. japonica. This work is the first to report about the mtCA of macroalgae and provides a basis for further analysis on seaweed CCM.