Identification and characterization of a carboxysomal y-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120

Date

2014

Authors

de Araujo, Charlotte
Arefeen, Dewan
Tadesse, Yohannes
Long, Benedict
Price, Graeme (Dean)
Rowlett, Roger S.
Kimber, Matthew S.
Espie, George S.

Journal Title

Journal ISSN

Volume Title

Publisher

Kluwer Academic Publishers

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 × 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.

Description

Keywords

Keywords: Nostoc; Nostoc sp.; Nostoc sp. PCC 7120; Thermosynechococcus elongatus; Thermosynechococcus elongatus BP-1 Carbonyl sulfide; Carboxysome; CcmM; Gamma-carbonic anhydrase; Nostoc sp. PCC 7120

Citation

Source

Photosynthesis Research

Type

Journal article

Book Title

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DOI

10.1007/s11120-014-0018-4

Restricted until

2037-12-31