A Multiprotein Bicarbonate Dehydration Complex Essential to Carboxysome Function in Cyanobacteria

Abstract

Carboxysomes are proteinaceous biochemical compartments that constitute the enzymatic "back end" of the cyanobacterial CO 2 -concentrating mechanism. These protein-bound organelles catalyze HCO 3 ؊ dehydration and photosynthetic CO 2 fixation. In Synechocystis sp. strain PCC6803 these reactions involve the ␤-class carbonic anhydrase (CA), CcaA, and Form 1B ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The surrounding shell is thought to be composed of proteins encoded by the ccmKLMN operon, although little is known about how structural and catalytic proteins integrate to form a functional carboxysome. Using biochemical activity assays and molecular approaches we have identified a catalytic, multiprotein HCO 3 ؊ dehydration complex (BDC) associated with the protein shell of Synechocystis carboxysomes. The complex was minimally composed of a CcmM73 trimer, CcaA dimer, and CcmN. Larger native complexes also contained RbcL, RbcS, and two or three immunologically identified smaller forms of CcmM (62, 52, and 36 kDa). Yeast two-hybrid analyses indicated that the BDC was associated with the carboxysome shell through CcmM73-specific protein interactions with CcmK and CcmL. Protein interactions between CcmM73 and CcaA, CcmM73 and CcmN, or CcmM73 and itself required the N-terminal ␥-CA-like domain of CcmM73. The specificity of the CcmM73-CcaA interaction provided both a mechanism to integrate CcaA into the fabric of the carboxysome shell and a means to recruit this enzyme to the BDC during carboxysome biogenesis. Functionally, CcaA was the catalytic core of the BDC. CcmM73 bound H 14 CO 3 ؊ but was unable to catalyze HCO 3 ؊ dehydration, suggesting that it may potentially regulate BDC activity.

Insights from multiple structures of the shell proteins from the β‐carboxysome

“…32 How the interior of the carboxysome is organized relative to the shell is understood only in rough terms. 23,24 Two recent studies have highlighted the role of the CcmM protein in the b-type carboxysome in binding several other carboxysome proteins, 28,29 including the CcmK shell proteins. 29 The structural details of these interactions have not been illuminated yet.…”

“…23,24 Two recent studies have highlighted the role of the CcmM protein in the b-type carboxysome in binding several other carboxysome proteins, 28,29 including the CcmK shell proteins. 29 The structural details of these interactions have not been illuminated yet. Knowing which regions of the shell proteins interact with other carboxysome proteins, including CcmM and possibly other proteins, would answer key functional questions.…”

“…Two independent studies on b-caboxysomes suggest that the CcmM protein plays a key organizing role. 28,29 In one study, 29 yeast two hybrid experiments showed direct interactions between CcmM and the CcmK (BMC-type) shell proteins under study in the present work. The atomic level details of those interactions have not been elucidated yet.…”

Metabolic and Transcriptomic Phenotyping of Inorganic Carbon Acclimation in the Cyanobacterium Synechococcus elongatus PCC 7942

“…Bicarbonate enters a special compartment, the carboxysome, where Rubisco is closely associated with the enzyme carbonic anhydrase, which converts bicarbonate into CO 2 . The action of carbonic anhydrase results in high C i inside the carboxysome, causing the saturation of Rubisco (Badger et al, 2006;Cot et al, 2008;Kaplan et al, 2008).…”

Carboxysomal carbonic anhydrases: Structure and role in microbial CO2 fixation

“…Examination of its predicted amino acid sequence tempts speculation that catalysis by its native form, if at all possible, may require complicated interaction(s) with multiple subunits and/or additional carboxysome components. Recombinant CcmM from Synechocystis PCC6803 is able to bind bicarbonate (or CO 2 ) [62], and a recent unpublished result (mentioned in [62]) suggests that carboxysome-enriched fractions from Nostoc PCC7120 have CA activity. Since this cyanobacterial species does not contain a ccaA homolog, perhaps CcmM provides the CA activity required for the function of the carboxysome as part of the CCM in that bacterium.…”