Identification and characterization of a gene encoding a vertebrate-type carbonic anhydrase in cyanobacteria

Abstract

A gene (designated ecaA) encoding a vertebrate-like (␣-type) carbonic anhydrase (CA) has been isolated from two disparate cyanobacteria, Anabaena sp. strain PCC 7120 and Synechococcus sp. strain PCC 7942. The deduced amino acid sequences correspond to proteins of 29 and 26 kDa, respectively, and revealed significant sequence similarity to human CAI and CAII, as well as Chlamydomonas CAHI, including conservation of most active-site residues identified in the animal enzymes. Structural similarities between the animal and cyanobacterial enzymes extend to the levels of antigenicity, as the Anabaena protein cross-reacts with antisera derived against chicken CAII. Expression of the cyanobacterial ecaA is regulated by CO 2 concentration and is highest in cells grown at elevated levels of CO 2 . Immunogold localization using an antibody derived against the ecaA protein indicated an extracellular location. Preliminary analysis of Synechococcus mutants in which ecaA has been inactivated by insertion of a drug resistance cassette suggests that extracellular carbonic anhydrase plays a role in inorganic-carbon accumulation by maintaining equilibrium levels of CO 2 and HCO 3 ؊ in the periplasm.Carbonic anhydrase (CA), a zinc metalloenzyme, catalyzes the reversible hydration of CO 2 and plays a significant role in such processes as pH homeostasis, respiratory gas exchange, photosynthesis, and ion transport (3,6,24). On the basis of distinct amino acid sequences (including active-site regions), three major CA groups have been described: (i) the ␣, or "eukaryotic," group, which includes CA isoforms found in vertebrates; (ii) the ␤, or "bacterial," group, which includes CA enzymes in eubacteria and structurally similar CA isoforms localized in the higher-plant chloroplast and cytosol; and (iii) a recently identified ␥, or "archaebacterial," group of CAs thought to play a role in acetate metabolism (1, 12). Each of these CA types is presumed to have evolved independently (12). In the cyanobacteria, a ␤-type CA has been identified previously and was shown to be required for photosynthesis at air levels of CO 2 (8). Efficient photosynthetic inorganic carbon (C i ) assimilation by cyanobacteria at these limiting levels of C i requires the operation of CO 2 -concentrating mechanisms (CCMs) (3,4,6,13). The activity of CCMs results in the energy-dependent transport of both HCO 3 Ϫ and CO 2 across the cytoplasmic membrane and the formation of a large intracellular pool of C i which is used during photosynthesis. Along with as yet unidentified CO 2 /HCO 3 Ϫ transporter proteins and energization components involving NADP(H) dehydrogenasedependent photosystem I electron flow (14, 19), a proteinencapsulated aggregate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) called a "carboxysome" is an integral part of a cyanobacterial CCM. It is hypothesized that associated with the cyanobacterial carboxysome is the ␤-type CA, a product of the icfA (also designated ccaA) gene (8,20,27). This CA exhibits significant amino acid sequence si...

Extracellular carbonic anhydrases of the stromatolite-forming cyanobacterium Microcoleus chthonoplastes

“…This is consistent with the evidence that only a few prokaryotic genomes encode a-class enzymes. However, a-CAs have been found in some bacteria (Nafi et al, 1990;Chirica et al, 1997) and cyanobacteria (Soltes-Rak et al, 1997;Dudoladova et al, 2004) where they have been characterized as extracellular enzymes, similar to the a-CA of M. chthonoplastes. Thus, the presence of this class of CAs in relict organisms, as well as in other bacteria, indicates that a-CAs are as ancient as b-and c-CAs.…”

A periplasmic, α-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake

“…palustris, and probably in other PNSB, is to accelerate the bicarbonate-CO # conversion in the periplasmic space. This process might be analogous to CA function in cyanobacteria and algae ; however, intact cells of cyanobacteria exhibit very low CA activity and inactivation of the cyanobacterial ecaA gene, which encodes extracellular CA, did not give a dramatic change in photosynthetic growth (Soltes-Rak et al, 1997). Although there are slight differences in periplasmic CA activities, CA seems to function universally in C i utilization in photosynthetic microorganisms.…”

Carbonic anhydrases in plants and algae

“…In photosynthetic organisms only a few a-CA have been identified at this time. One has been found in a cyanobacterium (Soltes-Rak, Mulligan & Coleman 1997). The green algae C. reinhardtii (Karlsson et al 1998) and Dunaliella salina (Fisher et al 1996;Yang, Zhang & Xu 1999) have three a-CAs.…”