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The N-terminal extension is essential for the formation of the active dimeric structure of liver peroxisomal alanine:glyoxylate aminotransferase.
[primary hyperoxaluria type 1]
Alanine
:
glyoxylate
aminotransferase
(
AGT
)
is
a
pyridoxal-phosphate
(
PLP
)
-
dependent
enzyme
.
Its
deficiency
causes
the
hereditary
kidney
stone
disease
primary
hyperoxaluria
type
1
.
AGT
is
a
highly
stable
compact
dimer
and
the
first
21
residues
of
each
subunit
form
an
extension
which
wraps
over
the
surface
of
the
neighboring
subunit
.
Naturally
occurring
and
artificial
amino
acid
replacements
in
this
extension
create
changes
in
the
functional
properties
of
AGT
in
mammalian
cells
,
including
relocation
of
the
enzyme
from
peroxisomes
to
mitochondria
.
In
order
to
elucidate
the
structural
and
functional
role
of
this
N-
terminal
extension
,
we
have
analyzed
the
consequences
of
its
removal
using
a
variety
of
biochemical
and
cell
biological
methods
.
When
expressed
in
Escherichia
coli
,
the
N-
terminal
deleted
form
of
AGT
showed
the
presence
of
the
protein
but
in
an
insoluble
form
resulting
in
only
a
10
%
soluble
yield
as
compared
to
the
full-length
version
.
The
purified
soluble
fraction
showed
reduced
affinity
for
PLP
and
greatly
reduced
catalytic
activity
.
Although
maintaining
a
dimer
form
,
it
was
highly
prone
to
self-aggregation
.
When
expressed
in
a
mammalian
cell
line
,
the
truncated
construct
was
normally
targeted
to
peroxisomes
,
where
it
formed
large
stable
but
catalytically
inactive
aggregates
.
These
results
suggest
that
the
N-
terminal
extension
plays
an
essential
role
in
allowing
AGT
to
attain
its
correct
conformation
and
functional
activity
.
The
precise
mechanism
of
this
effect
is
still
under
investigation
.