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

Abstract

Intact cells of the purple non-sulfur bacterium Rhodopseudomonas palustris growing anaerobically, but not aerobically, contain carbonic anhydrase (CA) activity. The native enzyme was purified S2000-fold to apparent homogeneity and found to be a dimer with an estimated molecular mass of 54 kDa and a subunit molecular mass of 27 kDa. The CA gene (acaP) was cloned and its sequence revealed that it was homologous to α-type CAs. The upstream region of acaP was fused to the lacZ gene and β-galactosidase activity was measured under different growth conditions. Acetazolamide inhibited purified CA with an IC 50 in the range of 10 N8 M, and in the culture media concentrations as low as 30 µM inhibited phototrophic growth under anaerobic, light conditions when bicarbonate was used. An acaP ::Kan r mutant strain was constructed by insertion of a kanamycin-resistance cassette and showed a growth pattern similar to wild-type cells grown in the presence of CA inhibitor. CO 2 gas supplied as an inorganic carbon source reversed the effect of mutation or acetazolamide. CA activity measurements, fusion and Western blot experiments confirmed that CA is expressed under different anaerobic conditions independently of bicarbonate or CO 2 and that there is no expression under aerobic conditions.

Characterization of carbonic anhydrase from diversified genus for biomimetic carbon-dioxide sequestration

“…5). Similar results have been reported for α-CA in H. pylori, R. palustris and C. reinhardtii [26,28,29]. On the basis of more than 80% inhibition with sulphanilamide and a molecular weight in the region of ~29 kDa, it can be predicted that the CA from all the four isolates evaluated during the present study is of α-type, however confi rmation has to be done through immunoblotting.…”

“…Similar results have been reported in H. pylori and bacteria from the karst areas [26,27], whereas only the periplasmic fraction revealed a signifi cant activity in A. hydrophila (1.56 EU/ml) and A. caviae (1.57 EU/ml). Similar results [25,28,29]. Periplasmic location of the enzyme is signifi cant in maintenance and regulation of pH and ionic concentration.…”

Characterization of a Mesorhizobium loti α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation

“…Representatives of all CA classes are widespread in prokaryotes, indicating the ancient origins of these enzymes (33). In prokaryotes, ␣-CAs have been characterized from several species, including the cyanobacteria Synechococcus and Anabaena (34) and the eubacteria Neisseria gonorrhoeae (5), Helicobacter pylori (6), and Rhodopseudomonas palustris (29).…”

“…cynT, the only can paralog in E. coli, is transcribed as part of the cyanase (cyn) operon and serves to provide sufficient levels of cellular HCO 3 Ϫ for growth with cyanate as the nitrogen source (15). In R. palustris, a periplasmic ␣-type CA has been proposed to be essential for bicarbonate uptake (29). Finally, a periplasmic ␣-type CA from H. pylori has been shown to be upregulated by low environmental pH, being essential for buffering the periplasmic acidic pH.…”

Crystallographic study of wild-type carbonic anhydrase αCA1 fromChlamydomonas reinhardtii

“…However, there are some exceptions. CA from the alga Chlamydomonas reinhardtii is known to be a dimer (Ishida et al, 1993;Kamo et al, 1990) and CA from the bacterium Rhodopseudomonas palustris (Puská s et al, 2000) and the human isozyme CA-XII (Whittington et al, 2001) are also dimeric, while the human isozyme CA-IX is tetrameric (Alterio et al, 2009). The latter three CAs are membrane-associated proteins.…”