A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell

Abstract

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, CO(2) 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 CO(2)-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 (epsilon-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 CO(2) necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle.

FIG. 1.

Carboxysomes from H. neapolitanus. (A) Transmission electron micrograph of an H. neapolitanus cell containing numerous polyhedral carboxysomes (indicated by arrowheads). (B) Purified, negatively stained intact carboxysomes. (C) Negatively stained carboxysome shells after freeze-thaw treatment. Bars, 100 nm.

FIG. 2.

CA activity associated with purified carboxysomes. (A) Measurements of ¹⁸O exchange activity by MS. Experiments were initiated by the addition of 400 μM K₂¹³C¹⁸O₃ to the reaction vessel containing 100 mM EPPS-NaOH (pH 8.0) and 20 mM MgSO₄. The relative concentrations of ¹³C¹⁸O₂ (m/z = 49), ¹³C¹⁸O¹⁶O (m/z = 47), and ¹³C¹⁶O₂ (m/z = 45) were monitored prior to and following the addition of purified carboxysomes (cbx; 120 μg of protein) from H. neapolitanus. The time course of the uncatalyzed isotopic exchange reaction in buffer alone is depicted by the dotted lines. (B) Electrophoretic separation of polypeptides from a homogeneous carboxysome preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were visualized following Coomassie staining. (C) CA and RuBisCO activities in fractions collected following carboxysome purification on sucrose density gradients. Partially purified carboxysomes were loaded onto a 10 to 50% sucrose density gradient and centrifuged. Fractions from the gradient were collected and assayed.

FIG. 3.

Identification of CsoS3 as a CA. (A) Schematic of the cso gene cluster from H. neapolitanus. Bar, 1 kb. (B) Measurements of ¹⁸O exchange in cell lysates (1.5 mg of protein) of E. coli expressing epitope-tagged CsoS3 and each of the other polypeptides encoded by the H. neapolitanus cso gene cluster. For clarity, only the m/z 45 traces are presented. (C) ¹⁸O exchange activity in bacterial extracts (0.8 mg of protein) containing T7-CsoS3 from Synechococcus strain WH8102 and P. marinus strains MED4 and MIT9313.

FIG. 4.

Association of CsoS3 with the carboxysome shell. (A) ¹⁸O exchange activity in intact (cbx) and broken carboxysome preparations. After freeze-thaw treatment of purified carboxysomes, pelletable (p) (71 μg of protein) and soluble (s) (49 μg of protein) fractions were assayed. (B) Coomassie staining of carboxysomal proteins and immunodetection of CsoS3 and CbbL. The faint, unassigned bands are incompletely denatured aggregates of shell proteins.

FIG. 5.

Measurements of CsoS3 activity. (A) Effect of EZ (500 μM) on CsoS3-catalyzed ¹⁸O exchange activity. (Inset) Graph of inhibition of CsoS3 by increasing concentrations of EZ. (B) Electrometric and MS measurements of CA activity. Recombinant, affinity-purified CsoS3 (38 μg) was derived from the IMPACT system. Electrometric assays were performed at 4°C for CsoS3 (1.75 μg), purified intact carboxysomes (100 μg), and bovine erythrocyte CA (1 μg). The value in parentheses represents the calculated specific activity of CsoS3, assuming that this protein constitutes 5% of the total carboxysomal protein. Alternatively, the activity of CsoS3 (6 to 20 μg) was determined at 30°C by MS as a percentage of the initial rate of m/z 45 appearance. Note that the solubility of EZ is greatly reduced at 4°C. DTT, dithiothreitol; NTA, nitrilotriacetic acid; DPA, dipicolinic acid.

FIG. 6.

Unrooted phylogenetic tree for csoS3 homologues. Nucleotide sequence alignments were performed with ClustalX, version 1.81, and used to construct a neighbor-joining tree with MEGA, version 2.1. Bootstrap values are displayed at nodes as percentages of 1,000 replicates.

FIG. 7.

Positions of candidate metal-binding residues in ɛ CAs and known zinc ligands of other CAs. (A) Alignment for a conserved portion of the CsoS3 polypeptides from H. neapolitanus (Hn), T. denitrificans (Td), A. ferrooxidans (Af), T. intermedia (Ti), Synechococcus sp. strain WH8102 (WH), and Prochlorococcus sp. strains CCMP1378 (CC), MED4 (MED), MIT9313 (MIT), and SS120 (SS). Amino acids conserved among all proteins are shaded. Potential zinc-binding residues are indicated by asterisks. (B) Zinc ligands (shaded) determined from the X-ray crystal structures for human CAII (α class), P. purpureum CA (β class), and Methanosarcina thermophila Cam (γ class). It must be pointed out that β- and γ-class CAs are multimeric enzymes with multiple Zn²⁺ binding sites. Amino acid numbering is indicated at the right of each sequence.