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Link to original content: http://www.ncbi.nlm.nih.gov/pubmed/17012396
Characterization of the carboxysomal carbonic anhydrase CsoSCA from Halothiobacillus neapolitanus - PubMed Skip to main page content
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. 2006 Dec;188(23):8087-94.
doi: 10.1128/JB.00990-06. Epub 2006 Sep 29.

Characterization of the carboxysomal carbonic anhydrase CsoSCA from Halothiobacillus neapolitanus

Sabine Heinhorst  1 , Eric B Williams, Fei Cai, C Daniel Murin, Jessup M Shively, Gordon C Cannon
Affiliations

Characterization of the carboxysomal carbonic anhydrase CsoSCA from Halothiobacillus neapolitanus

Sabine Heinhorst et al. J Bacteriol. 2006 Dec.

Abstract

In cyanobacteria and many chemolithotrophic bacteria, the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is sequestered into polyhedral protein bodies called carboxysomes. The carboxysome is believed to function as a microcompartment that enhances the catalytic efficacy of RubisCO by providing the enzyme with its substrate, CO(2), through the action of the shell protein CsoSCA, which is a novel carbonic anhydrase. In the work reported here, the biochemical properties of purified, recombinant CsoSCA were studied, and the catalytic characteristics of the carbonic anhydrase for the CO(2) hydration and bicarbonate dehydration reactions were compared with those of intact and ruptured carboxysomes. The low apparent catalytic rates measured for CsoSCA in intact carboxysomes suggest that the protein shell acts as a barrier for the CO(2) that has been produced by CsoSCA through directional dehydration of cytoplasmic bicarbonate. This CO(2) trap provides the sequestered RubisCO with ample substrate for efficient fixation and constitutes a means by which microcompartmentalization enhances the catalytic efficiency of this enzyme.

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Figures

FIG. 1.
FIG. 1.
Carboxysomes of H. neapolitanus. (A) Transmission electron micrograph of an H. neapolitanus cell containing carboxysomes. (B) Purified, negatively stained intact carboxysomes. (C) Negatively stained carboxysomes after rupture by freeze-thaw treatment. In all panels, the bar represents 100 nm.
FIG. 2.
FIG. 2.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel of heterologously expressed CsoSCA. Lanes: 1, 20 μg of cell extract from uninduced E. coli cells containing pProExCsoSCA; 2, 20 μg of cell extract of induced cells; 3, 2 μg of rCsoSCA protein eluted from a Ni2+ affinity column; 4, purified carboxysomes (from a separate gel).
FIG. 3.
FIG. 3.
pH dependence of CsoSCA kcat for CO2 hydration. Turnover numbers were calculated by fitting initial rates (8 to 15 independent determinations for each data point) to the Michaelis-Menten equation as described in Materials and Methods and were plotted as log(kcat) versus pH (○, rCsoSCA; □, broken carboxysomes; ⋄, intact carboxysomes). The buffer/indicator pairs and wavelengths used were MES/chlorophenol red (A574) at pH 5.5 to 6.8; MOPS/p-nitrophenol (A400) at pH 6.8 to 7.5; HEPES/phenol red (A557) at pH 7.5 to 8.0; and TAPS/m-cresol purple (A578) at pH 8.0 to 9.0.
FIG. 4.
FIG. 4.
Reaction progress of CO2 hydration with intact and ruptured carboxysomes. Samples of carboxysomes were identical, except that the ruptured sample had been subjected to one round of freeze-thaw treatment before the assay was performed as described in Materials and Methods. The amount of carboxysome protein used in these assays corresponded to 0.25 μM CsoSCA (□, intact carboxysomes; ▪, broken carboxysomes).
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