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
2
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.
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