A Novel Evolutionary Lineage of Carbonic Anhydrase (ε Class) Is a Component of the Carboxysome Shell

A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes. In cyanobacteria and many chemolithoautotrophic bacteria, CO2 fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes. These structures play an integral role in a cellular CO2-concentrating mechanism and are essential components for autotrophic growth. Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes. Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp. and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity. The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO2 necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle. Many bacteria contain polyhedral protein microcompartments that are bounded by a 3- to 4-nm shell composed of several highly conserved polypeptides. However, the enzyme content of these structures varies depending on metabolic function and the bacterial strain in which they occur. For example, microcompartments found in a Salmonella sp. grown on propanediol contain propanediol dehydratase, while similar microcompartments appearing in Escherichia coli grown on ethanolamine contain ammonia lyase (12, 16). The widespread occurrence of such polyhedral bodies has led to the suggestion that their protein shell provides a catalytic advantage to the enzyme within. The archetypal example of a prokaryotic microcompartment is the carboxysome, which is found in all cyanobacteria and many chemolithoautotrophic bacteria. Carboxysomes from the chemolithoautotroph Halothiobacillus (formerly Thiobacillus) neapolitanus measure 100 to 120 nm across and are filled with the CO2-fixing enzyme of the Calvin-Benson-Bassham cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, form 1A), which is essential for autotrophic carbon metabolism (7, 27, 34). In addition to the large (CbbL) and small (CbbS) subunits of RuBisCO, up to seven additional polypeptides are associated with the carboxysome; five of these have been identified as shell proteins (3, 4, 11). The carboxysome polypeptides are encoded by genes within the cso gene cluster, an apparent operon that also includes cbbL and cbbS (29). A similar organization of carboxysome genes has been reported for three other members of the former genus Thiobacillus (6)