Annual Report 2006

P
lant Physiology Department
The research activities of our department
are mainly focused on the elucidation of
molecular mechanisms of important physiological
processes in plants. The major topics in fiscal
2005 are as follows.
Photosynthesis and carbon metabolism
Identification of critical residues
for substrate recognition of
CtpA, an integral protease for
assembly of oxygen-evolving
complex of photosystem II.
Carboxyl-terminal processing protease A
(CtpA) is an indispensable protease for
assembling of the photosynthetic oxygen
evolving machinery, which performs
proteolytic processing of the precursor form of
the D1 protein, a pivotal subunit of photosystem
II. The CtpA is well known to have very
narrow substrate specificity, which is one of the
common properties of proteases involved in
regulation of cellular processes. In order to
approach molecular basis of the high specificity,
we examined each residue of the spinach CtpA
by several concepts that tendency of
conservation in the CtpA family, distance from
the catalytic center and accessibility of
substrates. I400, V416 and Y419 were extracted
as candidate residues involved in substrate
recognition of the protease (Fig. 1A).
We introduced a series of site-directed
mutations about these residues of the spinach
gene and transformed the mutagenized
genes into deleted sp. PCC
6803 cells, which helps rapid evaluation of
effects of each mutation onto the CtpA activity.
These analyses revealed that V416 and Y419
are not so important for the CtpA action,
because these positions could be accepted
various substitutions without deactivation of
the CtpA. In contrast, I400 is likely to be critical
for CtpA activity. It is noteworthy that I400T, a
relatively mild substitution, significantly
abolishes the activity and then
cells with the mutant gene could not grow on
Fig. 1 (A)
Three dimensional structure of the CtpA and positions
of our target residues
Catalytic center residues, S372 and K397 were
colored with red and blue, respectively. CtpA has two
grooves close to the catalytic center. I400, V416 and
Y419 (colored with orange) emerged on surface of the
narrow groove.
Fig. 1 (B)
Photoautotrophic growth test of the several mutants
cells carrying the I400T mutant
gene could not grow on photoautotrophic medium (-
glucose). In contrast, V416T cells did not abolish the
CtpA activity and then the mutant as well as WT cells
grew well on the same medium.