PhD Exit Seminar for Maryam Moazami-Goudarzi (Espie Lab)

The Structure, Function, Kinetics, Regulation, and Interactions of β-Carboxysomal Proteins CcmM and CcaA

 Abstract

Carboxysomes are proteinaceous biochemical micro-compartments essential for photosynthetic CO2 fixation in cyanobacteria. These self-assembling structures encapsulate Ribulose-1,5-bisphosphate carboxylase/oxygenase and a carbonic anhydrase (CA) creating a segregated environment where both enzyme and substrate CO2 are concentrated. CO2 is generated within the β-carboxysome by the catalyzed dehydration HCO3- mediated by two evolutionarily distinct lineages of CA, the β-class CcaA and the redox-regulated γ-class CcmM. Here we determined the components of the redox switching mechanism in CcmM from Nostoc sp. PCC7120 and provided a reaction mechanism through which the oxidation / reduction of Cys194 – Cys200 disulfide bond is transmitted to the active site to change the level of enzymatic activity between the carboxysome-localized and cytosol-localized enzyme. Switching between activity states in this trimeric protein involves residues from two adjacent protomers. Of profound importance are Pro9, Pro10, Thr11, and Trp13 from the AAPPTPWS motif and Tyr192 that forms part of a trans-protomer activation triad Tyr192’ – Asp55 – Glu56 within the active site. In a subset of cyanobacteria, however, we provide evidence that CcaA either complements or replaces CcmM as the carboxysomal CA, though the kinetic parameters, kcat and kcat / KM, are of similar magnitude. Notably, hexameric CcaA is targeted to the carboxysome through its pM binding affinity for trimeric CcmM, forming a symmetric CcmM3CcaA6CcmM3 complex. The tight association of these two structurally distinct but catalytically similar proteins has led to the evolution of distinct β-carboxysome sub-types in which CcmM is the sole CA (BDC1), CcmM and CcaA are co-contributors of CA activity (BDC2) and in which CcaA is the sole CA (BDC3). In the latter case, a catalytically inactive CcmM is retained as a structural scaffold. Though bioinformatics analysis and biochemical reconstitution experiments we demonstrate that CcmM from BDC3-strains have lost CA activity through the devolution of Cys194 – Cys200, Tyr192 and other residues of the redox activation domain. Rank ordering of the CcmM kinetic parameters within a multiple sequence alignment allowed for the identification of a minimal complement of amino acid residues required for CcmM CA activity, substitution of which leads to an enzyme in a low activity state or with no activity.

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Join Zoom Meeting

Wednesday, December 1st, 2021 at 1:10 pm

https://utoronto.zoom.us/j/89579915356

Meeting ID: 895 7991 5356

Host: George Espie (george.espie@utoronto.ca)

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