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: Charlotte de Araujo, Dewan Arefeen, Yohannes Tadesse, Benedict M Long, G Dean Price, Roger S Rowlett, Matthew S Kimber, George S Espie
• Journal: Photosynthesis research (Photosynth Res) Vol. 121 Issue 2-3 Pg. 135-50 (Sep 2014) ISSN: 1573-5079 [Electronic] Netherlands
• PMID: 24907906 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bacterial Proteins
• Carbon Dioxide
• Ribulose-Bisphosphate Carboxylase
• Carbonic Anhydrases

Topics

• Bacterial Proteins (metabolism)
• Carbon Dioxide (metabolism)
• Carbonic Anhydrases (chemistry, metabolism)
• Nostoc (enzymology)
• Ribulose-Bisphosphate Carboxylase (metabolism)