title: Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120.
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 × 10(4) s(-1) and k cat/K m of 4.1 × 10(6) 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.
authors.affiliation: Department of Cell & Systems Biology, University of Toronto, Mississauga, ON, Canada.
authors.forename: Charlotte
authors.initials: C
authors.lastname: de Araujo
authors.forename: Dewan
authors.initials: D
authors.lastname: Arefeen
authors.forename: Yohannes
authors.initials: Y
authors.lastname: Tadesse
authors.forename: Benedict M
authors.initials: BM
authors.lastname: Long
authors.forename: G Dean
authors.initials: GD
authors.lastname: Price
authors.forename: Roger S
authors.initials: RS
authors.lastname: Rowlett
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Kimber
authors.forename: George S
authors.initials: GS
authors.lastname: Espie
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism*; D046937:Nostoc / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002245:Carbon Dioxide; D012273:Ribulose-Bisphosphate Carboxylase; D002256:Carbonic Anhydrases