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Title: Structural basis of the oxidative activation of the carboxysomal [gamma]-carbonic anhydrase, CcmM

Abstract

Cyanobacterial RuBisCO is sequestered in large, icosahedral, protein-bounded microcompartments called carboxysomes. Bicarbonate is pumped into the cytosol, diffuses into the carboxysome through small pores in its shell, and is then converted to CO{sub 2} by carbonic anhydrase (CA) prior to fixation. Paradoxically, many {beta}-cyanobacteria, including Thermosynechococcus elongatus BP-1, lack the conventional carboxysomal {beta}-CA, ccaA. The N-terminal domain of the carboxysomal protein CcmM is homologous to {gamma}-CA from Methanosarcina thermophila (Cam) but recombinant CcmM derived from ccaA-containing cyanobacteria show no CA activity. We demonstrate here that either full length CcmM from T. elongatus, or a construct truncated after 209 residues (CcmM209), is active as a CA - the first catalytically active bacterial {gamma}-CA reported. The 2.0 {angstrom} structure of CcmM209 reveals a trimeric, left-handed {beta}-helix structure that closely resembles Cam, except that residues 198-207 form a third {alpha}-helix stabilized by an essential Cys194-Cys200 disulfide bond. Deleting residues 194-209 (CcmM193) results in an inactive protein whose 1.1 {angstrom} structure shows disordering of the N- and C-termini, and reorganization of the trimeric interface and active site. Under reducing conditions, CcmM209 is similarly partially disordered and inactive as a CA. CcmM protein in fresh E. coli cell extracts is inactive, implying that the cellularmore » reducing machinery can reduce and inactivate CcmM, while diamide, a thiol oxidizing agent, activates the enzyme. Thus, like membrane-bound eukaryotic cellular compartments, the {beta}-carboxysome appears to be able to maintain an oxidizing interior by precluding the entry of thioredoxin and other endogenous reducing agents.« less

Authors:
; ; ; ;  [1]
  1. Guelph
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE
OSTI Identifier:
1002286
Resource Type:
Journal Article
Journal Name:
Proc. Natl. Acad. Sci. USA
Additional Journal Information:
Journal Volume: 107; Journal Issue: (6) ; 02, 2010; Journal ID: ISSN 0027-8424
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; ACID CARBONATES; CARBONIC ANHYDRASE; COMPARTMENTS; CYANOBACTERIA; DISULFIDES; MACHINERY; OXIDIZERS; PROTEINS; REDUCING AGENTS; RESIDUES; THIOLS

Citation Formats

Peña, Kerry L, Castel, Stephane E, de Araujo, Charlotte, Espie, George S, Kimber, Matthew S, and Toronto). Structural basis of the oxidative activation of the carboxysomal [gamma]-carbonic anhydrase, CcmM. United States: N. p., 2010. Web. doi:10.1073/pnas.0910866107.
Peña, Kerry L, Castel, Stephane E, de Araujo, Charlotte, Espie, George S, Kimber, Matthew S, & Toronto). Structural basis of the oxidative activation of the carboxysomal [gamma]-carbonic anhydrase, CcmM. United States. https://doi.org/10.1073/pnas.0910866107
Peña, Kerry L, Castel, Stephane E, de Araujo, Charlotte, Espie, George S, Kimber, Matthew S, and Toronto). 2010. "Structural basis of the oxidative activation of the carboxysomal [gamma]-carbonic anhydrase, CcmM". United States. https://doi.org/10.1073/pnas.0910866107.
@article{osti_1002286,
title = {Structural basis of the oxidative activation of the carboxysomal [gamma]-carbonic anhydrase, CcmM},
author = {Peña, Kerry L and Castel, Stephane E and de Araujo, Charlotte and Espie, George S and Kimber, Matthew S and Toronto)},
abstractNote = {Cyanobacterial RuBisCO is sequestered in large, icosahedral, protein-bounded microcompartments called carboxysomes. Bicarbonate is pumped into the cytosol, diffuses into the carboxysome through small pores in its shell, and is then converted to CO{sub 2} by carbonic anhydrase (CA) prior to fixation. Paradoxically, many {beta}-cyanobacteria, including Thermosynechococcus elongatus BP-1, lack the conventional carboxysomal {beta}-CA, ccaA. The N-terminal domain of the carboxysomal protein CcmM is homologous to {gamma}-CA from Methanosarcina thermophila (Cam) but recombinant CcmM derived from ccaA-containing cyanobacteria show no CA activity. We demonstrate here that either full length CcmM from T. elongatus, or a construct truncated after 209 residues (CcmM209), is active as a CA - the first catalytically active bacterial {gamma}-CA reported. The 2.0 {angstrom} structure of CcmM209 reveals a trimeric, left-handed {beta}-helix structure that closely resembles Cam, except that residues 198-207 form a third {alpha}-helix stabilized by an essential Cys194-Cys200 disulfide bond. Deleting residues 194-209 (CcmM193) results in an inactive protein whose 1.1 {angstrom} structure shows disordering of the N- and C-termini, and reorganization of the trimeric interface and active site. Under reducing conditions, CcmM209 is similarly partially disordered and inactive as a CA. CcmM protein in fresh E. coli cell extracts is inactive, implying that the cellular reducing machinery can reduce and inactivate CcmM, while diamide, a thiol oxidizing agent, activates the enzyme. Thus, like membrane-bound eukaryotic cellular compartments, the {beta}-carboxysome appears to be able to maintain an oxidizing interior by precluding the entry of thioredoxin and other endogenous reducing agents.},
doi = {10.1073/pnas.0910866107},
url = {https://www.osti.gov/biblio/1002286}, journal = {Proc. Natl. Acad. Sci. USA},
issn = {0027-8424},
number = (6) ; 02, 2010,
volume = 107,
place = {United States},
year = {Mon Apr 26 00:00:00 EDT 2010},
month = {Mon Apr 26 00:00:00 EDT 2010}
}