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

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, includingThermosynechococcus elongatus BP-1, lack the conventional carboxysomal β-CA,ccaA . The N-terminal domain of the carboxysomal protein CcmM is homologous to γ-CA fromMethanosarcina thermophila (Cam) but recombinant CcmM derived fromccaA -containing cyanobacteria show no CA activity. We demonstrate here that either full length CcmM fromT. 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 freshE. 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.