Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120 |
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Authors: | Charlotte de Araujo Dewan Arefeen Yohannes Tadesse Benedict M Long G Dean Price Roger S Rowlett Matthew S Kimber George S Espie |
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Institution: | 1. Department of Cell & Systems Biology, University of Toronto, Mississauga, ON, Canada 2. Department of Biology, University of Toronto, 3359 Mississauga Road, Mississauga, ON, L5L 1C6, Canada 3. Division of Plant Science, Research School of Biology, Australian National University, Canberra, ACT, Australia 4. Department of Chemistry, Colgate University, Hamilton, NY, USA 5. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada
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Abstract: | Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO3 ? dehydration and the subsequent fixation of CO2. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein–protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k cat of 2.0 × 104 s?1 and k cat/K m of 4.1 × 106 M?1 s?1 at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25–35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO2 analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes. |
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