Structural basis of the oxidative activation of the carboxysomal γ-carbonic anhydrase, CcmM

• Published in: Proceedings of the National Academy of Sciences - PNAS 2010-02, Vol.107 (6), p.2455-2460
• Main Authors: Peña, Kerry L, Castel, Stephane E, de Araujo, Charlotte, Espie, George S, Kimber, Matthew S
• Format: Article
• Language: English
• Subjects: Active sites; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites - genetics; Biological Sciences; Carbon dioxide; Carbon Dioxide - chemistry; Carbon Dioxide - metabolism; Carbonic Anhydrase Inhibitors - pharmacology; Carbonic Anhydrases - chemistry; Carbonic Anhydrases - genetics; Carbonic Anhydrases - metabolism; Catalysis - drug effects; Crystallography, X-Ray; Crystals; Cyanobacteria; Cyanobacteria - enzymology; Cyanobacteria - genetics; Cytoplasmic Granules - enzymology; Cytosol; Disulfides; Disulfides - chemistry; Disulfides - metabolism; Enzymes; Ethoxzolamide - pharmacology; Ferries; Models, Molecular; Molecular Sequence Data; Mutation; Oxidation-Reduction; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Protein subunits; Proteins; Sequence Homology, Amino Acid
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0910866107

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 CO₂ by carbonic anhydrase (CA) prior to fixation. Paradoxically, many β-cyanobacteria, including Thermosynechococcus elongatus BP-1, lack the conventional carboxysomal β-CA, ccaA. The N-terminal domain of the carboxysomal protein CcmM is homologous to γ-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 γ-CA reported. The 2.0 Å structure of CcmM209 reveals a trimeric, left-handed β-helix structure that closely resembles Cam, except that residues 198-207 form a third α-helix stabilized by an essential Cys194-Cys200 disulfide bond. Deleting residues 194-209 (CcmM193) results in an inactive protein whose 1.1 Å 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 β-carboxysome appears to be able to maintain an oxidizing interior by precluding the entry of thioredoxin and other endogenous reducing agents.