Characterization of a novel type of carbonic anhydrase that acts without metal cofactors

Abstract

Background: Carbonic anhydrases (CAs) are universal metalloenzymes that catalyze the reversible conversion of carbon dioxide (CO₂) and bicarbonate (HCO₃^-). They are involved in various biological processes, including pH control, respiration, and photosynthesis. To date, eight evolutionarily unrelated classes of CA families (α, β, γ, δ, ζ, η, θ, and ι) have been identified. All are characterized by an active site accommodating the binding of a metal cofactor, which is assumed to play a central role in catalysis. This feature is thought to be the result of convergent evolution.

Results: Here, we report that a previously uncharacterized protein group, named "COG4337," constitutes metal-independent CAs from the newly discovered ι-class. Genes coding for COG4337 proteins are found in various bacteria and photosynthetic eukaryotic algae. Biochemical assays demonstrated that recombinant COG4337 proteins from a cyanobacterium (Anabaena sp. PCC7120) and a chlorarachniophyte alga (Bigelowiella natans) accelerated CO₂ hydration. Unexpectedly, these proteins exhibited their activity under metal-free conditions. Based on X-ray crystallography and point mutation analysis, we identified a metal-free active site within the cone-shaped α+β barrel structure. Furthermore, subcellular localization experiments revealed that COG4337 proteins are targeted into plastids and mitochondria of B. natans, implicating their involvement in CO₂ metabolism in these organelles.

Conclusions: COG4337 proteins shared a short sequence motif and overall structure with ι-class CAs, whereas they were characterized by metal independence, unlike any known CAs. Therefore, COG4337 proteins could be treated as a variant type of ι-class CAs. Our findings suggested that this novel type of ι-CAs can function even in metal-poor environments (e.g., the open ocean) without competition with other metalloproteins for trace metals. Considering the widespread prevalence of ι-CAs across microalgae, this class of CAs may play a role in the global carbon cycle.

Keywords: Carbonic anhydrase; Chlorarachniophytes; Convergent evolution; Crystal structure; Cyanobacteria; Metal cofactor; Microalgae; Plastid.

Fig. 1

CA activity of COG4337 proteins. a Schematic images of Bn86287 and all2909 proteins. b Sequence alignment of COG4337 domains extracted from Bn86287 and all2909. Amino acids conserved in all and three of the four domains are shaded in black and gray, respectively. Positions of α-helices and β-strands estimated by X-ray crystallography are shown above the alignment. c–e CO₂ hydration activity of Bn86287, all2909, and α-class bovine CA (BCA) under various conditions: +EDTA, proteins were treated with 50 mM EDTA and 6 M urea; +PDA, proteins were treated with 50 mM 2,6-pyridinedicarboxylic acid (PDA); chemical symbols, metal ions were added to protein solution; I⁻, 1 mM KI was added to the reaction solution as an inhibitor. Bovine serum albumin (BSA) was used as a negative control. Significant differences compared to non-treated samples were determined by the two-tailed Student’s t test (*P < 0.02, **P < 0.01). In the graphs, error bars represent the SD calculated from three individual experiments. f HCO₃ dehydration activity of COG4337 proteins and BCA

Fig. 2

Structures of COG4337 proteins. a, b Dimeric structures of Bn86287 and all2909 are displayed as a ribbon diagram. A bicarbonate molecule (yellow and red spheres) is located inside the cone-shaped barrel. c Structural alignment of the four COG4337 domains. d Cross-sections of all2909 represent a bent finger-like cavity (transparent gay) containing a bicarbonate (BCT) and residues lining the cavity. e, f Active site overlays of the four COG4337 domains with bicarbonate (BCT) and iodide ion (IOD), respectively. As a difference, the position of Lys180 in all2909 is occupied by Tyr224/388/552 in Bn86287. g Dimeric structure of an uncharacterized protein of Xanthomonas campestris (PDB ID: 3H51). All images were prepared with PyMOL v. 2.3.3 (Schrödinger)

Fig. 3

Point mutations of active site residues. a, b Active sites of all2909 and the 3rd domain of Bn86287. Simulated annealing F_o–F_c omit maps (green) for bicarbonate (BCT) are displayed at a contour level of 3.0 σ. Relevant distances between bicarbonate oxygen atoms/iodide (IOD) and neighbor residues are indicated by dashed lines with numbers. The images were prepared with PyMOL v. 2.3.3 (Schrödinger). c, d CO₂ hydration activity of all2909 and the 3rd domain of Bn86287 and their mutants. Mutated residues are indicated by the one-letter code and number. Error bars indicate the SD of triplicate experiments. Significant differences compared to the WT were determined by the two-tailed Student’s t test and indicated by asterisks (P < 0.005)

Fig. 4

Subcellular localization of Bn86287 and Bn50950. a Immunoblot analysis of an anti-Bn86287 antibody against total proteins of Bigelowiella natans. A mature form of Bn86287 is estimated to be 55–59 kDa. b Confocal images of B. natans cells labeling with the anti-Bn86287 and an FITC-conjugated secondary antibody. Chlorophyll autofluorescence (Chl) is shown in red. c Immungold localization of Bn86287 in the plastid stroma of B. natans. Mt, mitochondrion; N, nucleus; Nm, nucleomorph; P, plastid; Py, pyrenoid. d Confocal images of the chlorarachniophyte Amorphochlora amoebiformis expressing the YFP fusion protein of mitochondrial Bn50950 (upper), and a cell stained by MitoTracker Orange CMTMRos (lower)