Sequence analysis of the carboxysomal carbonic anhydrase (CcaA) from
Synechocystis PCC6803,
Synechococcus PCC7942 and Nostoc
ATCC29133, indicated high sequence identity to the β class of plant and
bacterial carbonic anhydrases (CA), and conservation of the active site
region. However, the cyanobacterial enzyme has a
C-terminal extension of about 75 amino acids (aa) not
found in the plant enzymes, and largely absent from other bacterial enzymes.
Using recombinant DNA technology, genes encoding
C-terminal truncation products of up to 127 aa were
overexpressed in E. coli, and partially purified lysates
were analysed for CA-mediated exchange of 18O between
13C18O2and
H216O. Recombinant CcaA proteins
with up to 60 aa removed (CcaAΔ60) were catalytically competent, but
beyond this there was an abrupt loss of activity. CcaAΔ0, along with
CcaAΔ40 and CcaAΔ60, also catalysed the hydrolysis of carbon
oxysulfide (COS; an isoelectronic structural analogue of
CO2), but CcaAΔ63 and CcaAΔ127 did not,
indicating that truncations greater than 62 aa resulted in a general loss of
catalytic competency. Analysis of protein-protein interaction using the yeast
two-hybrid system revealed that CcaA did not interact with the large or small
Rubisco subunits (RbcL and RbcS, respectively) of
Synechocystis, but there was strong CcaA-CcaA
interaction. This protein interaction also ceased with
C-terminal truncations in CcaA greater than 60 aa. The
correlation between loss of CcaA-CcaA interaction and CcaA catalytic activity
suggests that the proximal portion of the C-terminal extension is required for
oligomerization, and that this oligomerization is essential for catalysis by
the cyanobacterial enzyme. Thus, the C-terminal extension may play an
important role in the function of CA within cyanobacterial carboxysomes, which
is not required by the higher plant enzymes.
Enzymatic Characteristics of Higher Plant Carbonic Anhydrase and Its Role in Photosynthesis