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.
| Original language | English |
|---|---|
| Pages (from-to) | 135-150 |
| Number of pages | 16 |
| Journal | Photosynthesis Research |
| Volume | 121 |
| Issue number | 2-3 |
| DOIs | |
| Publication status | Published - Sept 2014 |
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De Araujo, C., Arefeen, D., Tadesse, Y., Long, B. M., Price, G. D., Rowlett, R. S., Kimber, M. S., & Espie, G. S. (2014). Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120. Photosynthesis Research, 121(2-3), 135-150. https://doi.org/10.1007/s11120-014-0018-4
De Araujo, Charlotte ; Arefeen, Dewan ; Tadesse, Yohannes et al. / Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120. In: Photosynthesis Research. 2014 ; Vol. 121, No. 2-3. pp. 135-150.
@article{5aa382e3b1544ae284c084444b74cc41,
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 × 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.",
keywords = "Carbonyl sulfide, Carboxysome, CcmM, Gamma-carbonic anhydrase, Nostoc sp. PCC 7120",
author = "{De Araujo}, Charlotte and Dewan Arefeen and Yohannes Tadesse and Long, {Benedict M.} and Price, {G. Dean} and Rowlett, {Roger S.} and Kimber, {Matthew S.} and Espie, {George S.}",
year = "2014",
month = sep,
doi = "10.1007/s11120-014-0018-4",
language = "English",
volume = "121",
pages = "135--150",
journal = "Photosynthesis Research",
issn = "0166-8595",
publisher = "Springer Netherlands",
number = "2-3",
}
De Araujo, C, Arefeen, D, Tadesse, Y, Long, BM, Price, GD, Rowlett, RS, Kimber, MS & Espie, GS 2014, 'Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120', Photosynthesis Research, vol. 121, no. 2-3, pp. 135-150. https://doi.org/10.1007/s11120-014-0018-4
Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120. / De Araujo, Charlotte; Arefeen, Dewan; Tadesse, Yohannes et al.
In: Photosynthesis Research, Vol. 121, No. 2-3, 09.2014, p. 135-150.
Research output: Contribution to journal › Article › peer-review
TY - JOUR
T1 - Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120
AU - De Araujo, Charlotte
AU - Arefeen, Dewan
AU - Tadesse, Yohannes
AU - Long, Benedict M.
AU - Price, G. Dean
AU - Rowlett, Roger S.
AU - Kimber, Matthew S.
AU - Espie, George S.
PY - 2014/9
Y1 - 2014/9
N2 - 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.
AB - 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.
KW - Carbonyl sulfide
KW - Carboxysome
KW - CcmM
KW - Gamma-carbonic anhydrase
KW - Nostoc sp. PCC 7120
UR - http://www.scopus.com/inward/record.url?scp=84904747085&partnerID=8YFLogxK
U2 - 10.1007/s11120-014-0018-4
DO - 10.1007/s11120-014-0018-4
M3 - Article
SN - 0166-8595
VL - 121
SP - 135
EP - 150
JO - Photosynthesis Research
JF - Photosynthesis Research
IS - 2-3
ER -
De Araujo C, Arefeen D, Tadesse Y, Long BM, Price GD, Rowlett RS et al. Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120. Photosynthesis Research. 2014 Sept;121(2-3):135-150. doi: 10.1007/s11120-014-0018-4
