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Putrescine — α-Ketoglutarate transaminase in E . coli
BIOCHEMICAL
Vol. 9, No. 2, 1962
AND BIOPHYSICAL
- a-KETOGLUTARATE
PUTRESCINE
IN
Ki-han
E. -
Kim
Received
August
has
polyamines earlier
of
to
to
oxidase.
We sole
that
fluorescens
but
Materials
putrescine
N
--coli
characteristics
enzymatic
the
sole
oxidized
a mutant
similar
manner
as
briefly
can
semialdehyde.
grow
However,
the
first
not
catalyzed
is for
this
a diamine
on putrescine
actively
with
evidence
reported
putrescine
by
reported
Y -aminobutyraldehyde,
was
first
adapted
neutralized
it
grown
on
was
found
the of
with
convert
Pseudomonas
new
step,
the by
a diamine
transaminase
and
here.
grow
HCl)
putrescine
condition this
to
mutant
that of
as the
on
source
the
sole
source grown (The
reported
99
sole
the
putrescine
be
and
as
“adaptation”. will
glucose
inorganic of of
cells evidence
elsewhere.)
al.,
fluorescens
succinic
mutant
et.
This
convert
and
this
the
the
putrescine.
could
B which
of
little
Pseudomonas
N source
of
1959).
The
are
of
--coli
extracts
Fredericks, to
degradation
contrast,
Y -aminobutyraldehyde
of -E.
Cell-free
in
to
(Mora,
By
relatively
y -aminobutyrate was
source.
1961.)
that
role
T phages.
received
showed C and
physiological
the
et.al., has
who
the
Methods
then
under
as
a transaminase,
B
Subsequently, selected
Fredericks
and
(O-2%, were
and
and
Tabor,
bacteria
the
obtained
properties
and
-E.
on
putrescine by
of its
by
on
bacteria
by
report
succinate
of
oxidase,
cells
now
(Jakoby
conversion
in
reviewed
putrescine
have
-to
recently
y -aminobutyraldehyde,
C and
putrescine
interest
on putrescine
via
assumed
some
one
grow
succinate
the
Chemistry University Michigan
spermidine
been
of Jakoby
It was
as
Tchen
of
polyamines
only
work
adapted
has
these
with the
T.
T.
State 2,
considerable and
work
degradation
was
been
putrescine
attention
COLI.
8, 1962
There
1962;
TRANSAMINASE
and
Department Wayne Detroit
RESEARCH COMMUNlCATIONS
salts
N.
both
with
These C and
constituted
adapted N. a mutant
for
this
and
All
work
to
the be
BIOCHEMICAL
Vol. 9, No. 2, 1962
reported
here
were
neutralized
with
HCl)
Freshly at
O°C
in
(O.lM,
a VirTis
The
has
will
amounts
1961).
no
on
Jakoby
the
reaction
and
both
this
and
a-ketoglutarate
yellow
purification
sulfate
fractionation, is
was
has
in
a pH
inhibited
13-alanine.
still
and
can
at
coupling
435mp
reaction
(Holmstedt,
mixture
by
paper
as
1951),
was
but
synthesized
was
it
chromatography
Primosigh,
putrescine
much
lower mutant
inorganic contained
incubated If
formed.
was according
purchased
from
also
required
stimulated
by protamine
be
replaced
7 -aminoformation
of
putrescine
phosphate. sulfate
for
of
After
treatment
pyridoxal
included
The
presence
pyridoxal
and
and
ammonium
phosphate
was
I.
optimum 5 x 10
putrescine
between
obtained. the
requirement
by
with
was
product
glutamate
by
8-9
o-aminobenzaldehyde
was
Table
of
at pH
condensation
of
9 and
-4
M,
is
almost
completely
semicarbazide,
by
or
by
at pH or
1, 7-diaminoheptane,
Y -aminobutanol,
a-ketoglutarate
inactive
hydroxylamine
cadaverine
spermidine,
Substitution
this
was
enzyme
1, 3-diaminopropane,
and
Slotta
absolute
shown
Putrescine
glucose
al.,
yellow
and
an
enzyme
in
identified
o-aminobenzaldehyde
of the
completely
When
cells
the
pigment
This
observed.
resulted
was
was
and
partial
0~
Glutamate
supernatant
mixture,
butyraldehyde
by
the
minutes
by
at
Y -aminobutyraldehyde
glutamate
cyanide.
to
T-labeled
40
supernatant.
absorption
directly
activity
Corporation.
crude
a-ketoglutarate,
The
the
for
determined
added
(1959).
transaminase
crude
were
1944;
buffer
The
as
10 minutes
Discussion
When
not
et.
Nuclear
and
It is
enzyme.
Fredericks
England
the
was
quantitatively. and
Results
to hereafter
(0.2$,
109/m1.
phosphate
centrifuging
Y -aminobutyraldehyde
reagent
of for
beads.
after
measuring
putrescine
density
of
glass
supernatant
on
homogenized
parts
fine
referred
(Consden,
determined
grown
were
ten
of
and
the
systems
New
in
be
of
This
effect
two
not to
This
the
o-aminobenzaldehyde
et. al.,
in
with
RESEARCH COMMUNICATIONS
to a cell
cells
parts
in
mutant
salts
frozen
twenty
exclusively
this
inorganic or
and
x g.
with
and
with
homogenizer
7.0)
found
144,000
out
harvested
pH
was
carried
AND BIOPHYSICAL
but
histamine,
oxalacetate
or
tyramine
pyruvate
activity. was
cultured
salt
(5
approximately
for
successive
many
generations
transfers 30%
100
as
much
of
in one
transaminase
drop
a medium into as
of 50 ml)
did
cells
the
7.
BIOCHEMICAL
Vol. 9, No. 2, 1962
AND BIOPHYSICAL
TABLE
Enzyme
Used
Pyridoxal
Phosphate
Requirement
Pyridoxal
Phosphate
AOD protein
at
435mp per mg per 10 minutes
t
0 0
Mutant crude supernatent
t
. 094 .064
Mutant fraction
t
.283 0
grown
on
putrescine.
mixture contained 0.1 ml of crude supernatant or partially purified enzyme, 15 pmoles of putrescine, 200 pmoles of Tris buffer pH9.0, 10 p.moles of oThe mutant 20 yof pyridoxal phosphate when used. protamine sulfate and ammonium sulfate treatment. later.
It thus
appears
that
the
transaminase
is
constitutive
in
mutant. The
--E.
coli
1959)
subsequent is
and
the
catabolism
same
consists
as
of
found
of the
in
succinic
The
first
reaction
the
DPN’ was
reaction.
were
incubated
obtained
supernatant
after
‘7
second
by
Thus, with a lag
when the
of
no
t
reduction
of
demonstrated
Fredericks,
six glutamate of
101
DPN’.
t glutamate
DPN+ by
as
by
the well
y -amino-
formation as
by
of coupling
7 -aminobutyrate
supernatant,
or
and
DPNH
a-ketoglutarate
approximately
reduction
semialdehyde
a-ketoglutarate,
crude
7 -aminobutyrate gave
the
was and
of
t DPNH
succinic
--t succinate
reaction
strain
(Jakoby
-aminobutyrate
*
demonstrated
y-aminobutyrate
third
a-ketoglutarate, crude
was The
from
t DPN+
this
reactions:
a-ketoglutarate
semialdehyde
butyraldehyde. glutamate
t
in
fluorescens
three
t DPN’
y -aminobutyrate
y -aminobutyraldehyde
Pseudomonas
following
y-aminobutyraldehyde
to
I
E. coli B crude supernatant
The 1 ml of reaction corresponding amount of 4pmoles of a-ketoglutarate, aminobenzaldehyde, and fraction was obtained after Details will be reported
this
RESEARCH COMMUNICATIONS
a rapid minutes. alone
and
reduction Incubation
with
DPN’
of
DPN’
of and
the
BIOCHEMICAL
Vol. 9, No. 2, 1962
The three
conversion
reactions
putrescine and
of putrescine
listed
Experimentally,
above
a DPN+
to
should
and
to T-labeled
C)terell,
AND BIOPHYSICAL
1947) was
y -aminobutyraldehyde lead
readily
followed
to the formation
a-ketoglutarate
succinate
RESEARCH COMMUNICATIONS
(identified
of succinate.
dependent
conversion
by paper
chromatography,
demonstrated
using
by the
dialyzed
of T-labeled Lugg
crude
supernatant.
Summary
A mutant
of _E. --coli B was obtained which can grow on putrescine C and N source. This mutant converts putrescine to succinate
sole
y -aminobutyraldehyde, manner was
y -aminobutyrate
as found
in Pseudomonas
not catalyzed
constitutive
strain
oxidase,
of --E.
via
semialdehyde
The first
fluorescens.
by a diamine
in this
and succinic
as the
in similar
reaction,
but by a transaminase
however, which
is
coli.
Acknowledgment The
work
was
supported
by U. S.P.
H. S. grants
(H-4139)
and (A-5384).
References 1.
Consden,
R.,
2.
Holmstedt,
3.
Jakoby,
4.
Lugg,
J.W.H.,
5.
Mora,
P. T.,
Young,
6.
Slotta,
K.H.,
and Primosigh,
7.
Tabor,
H.,
W.
Gordon,
A.H.,
B.,
Lasson,
L.,
B.,
and Fredericks, and Overell,
Tabor,
B.G.,
C. W.,
and Martin, and Tham, J., B. T.,
Biochem.
R.,
Biochim.
J. Biol.
Chem.,
Nature,
and Rizivi, J.,
A. J.P.,
S.,
Nature,
and Rosenthal,
102
160,
Biophys. 234,
-38, Acta,
224 (1944). 48,
182 (1961).
2145 (1959).
87 (1947).
J. Bio. 168,
J.,
Chem.,
237,
157 (1962).
696 (1951).
S. M.,
Ann.
Rev.
Biochem.,
-30,
579 (1961)
Vol. 9, No. 2, 1962
AND BIOPHYSICAL
- a-KETOGLUTARATE
PUTRESCINE
IN
Ki-han
E. -
Kim
Received
August
has
polyamines earlier
of
to
to
oxidase.
We sole
that
fluorescens
but
Materials
putrescine
N
--coli
characteristics
enzymatic
the
sole
oxidized
a mutant
similar
manner
as
briefly
can
semialdehyde.
grow
However,
the
first
not
catalyzed
is for
this
a diamine
on putrescine
actively
with
evidence
reported
putrescine
by
reported
Y -aminobutyraldehyde,
was
first
adapted
neutralized
it
grown
on
was
found
the of
with
convert
Pseudomonas
new
step,
the by
a diamine
transaminase
and
here.
grow
HCl)
putrescine
condition this
to
mutant
that of
as the
on
source
the
sole
source grown (The
reported
99
sole
the
putrescine
be
and
as
“adaptation”. will
glucose
inorganic of of
cells evidence
elsewhere.)
al.,
fluorescens
succinic
mutant
et.
This
convert
and
this
the
the
putrescine.
could
B which
of
little
Pseudomonas
N source
of
1959).
The
are
of
--coli
extracts
Fredericks, to
degradation
contrast,
Y -aminobutyraldehyde
of -E.
Cell-free
in
to
(Mora,
By
relatively
y -aminobutyrate was
source.
1961.)
that
role
T phages.
received
showed C and
physiological
the
et.al., has
who
the
Methods
then
under
as
a transaminase,
B
Subsequently, selected
Fredericks
and
(O-2%, were
and
and
Tabor,
bacteria
the
obtained
properties
and
-E.
on
putrescine by
of its
by
on
bacteria
by
report
succinate
of
oxidase,
cells
now
(Jakoby
conversion
in
reviewed
putrescine
have
-to
recently
y -aminobutyraldehyde,
C and
putrescine
interest
on putrescine
via
assumed
some
one
grow
succinate
the
Chemistry University Michigan
spermidine
been
of Jakoby
It was
as
Tchen
of
polyamines
only
work
adapted
has
these
with the
T.
T.
State 2,
considerable and
work
degradation
was
been
putrescine
attention
COLI.
8, 1962
There
1962;
TRANSAMINASE
and
Department Wayne Detroit
RESEARCH COMMUNlCATIONS
salts
N.
both
with
These C and
constituted
adapted N. a mutant
for
this
and
All
work
to
the be
BIOCHEMICAL
Vol. 9, No. 2, 1962
reported
here
were
neutralized
with
HCl)
Freshly at
O°C
in
(O.lM,
a VirTis
The
has
will
amounts
1961).
no
on
Jakoby
the
reaction
and
both
this
and
a-ketoglutarate
yellow
purification
sulfate
fractionation, is
was
has
in
a pH
inhibited
13-alanine.
still
and
can
at
coupling
435mp
reaction
(Holmstedt,
mixture
by
paper
as
1951),
was
but
synthesized
was
it
chromatography
Primosigh,
putrescine
much
lower mutant
inorganic contained
incubated If
formed.
was according
purchased
from
also
required
stimulated
by protamine
be
replaced
7 -aminoformation
of
putrescine
phosphate. sulfate
for
of
After
treatment
pyridoxal
included
The
presence
pyridoxal
and
and
ammonium
phosphate
was
I.
optimum 5 x 10
putrescine
between
obtained. the
requirement
by
with
was
product
glutamate
by
8-9
o-aminobenzaldehyde
was
Table
of
at pH
condensation
of
9 and
-4
M,
is
almost
completely
semicarbazide,
by
or
by
at pH or
1, 7-diaminoheptane,
Y -aminobutanol,
a-ketoglutarate
inactive
hydroxylamine
cadaverine
spermidine,
Substitution
this
was
enzyme
1, 3-diaminopropane,
and
Slotta
absolute
shown
Putrescine
glucose
al.,
yellow
and
an
enzyme
in
identified
o-aminobenzaldehyde
of the
completely
When
cells
the
pigment
This
observed.
resulted
was
was
and
partial
0~
Glutamate
supernatant
mixture,
butyraldehyde
by
the
minutes
by
at
Y -aminobutyraldehyde
glutamate
cyanide.
to
T-labeled
40
supernatant.
absorption
directly
activity
Corporation.
crude
a-ketoglutarate,
The
the
for
determined
added
(1959).
transaminase
crude
were
1944;
buffer
The
as
10 minutes
Discussion
When
not
et.
Nuclear
and
It is
enzyme.
Fredericks
England
the
was
quantitatively. and
Results
to hereafter
(0.2$,
109/m1.
phosphate
centrifuging
Y -aminobutyraldehyde
reagent
of for
beads.
after
measuring
putrescine
density
of
glass
supernatant
on
homogenized
parts
fine
referred
(Consden,
determined
grown
were
ten
of
and
the
systems
New
in
be
of
This
effect
two
not to
This
the
o-aminobenzaldehyde
et. al.,
in
with
RESEARCH COMMUNICATIONS
to a cell
cells
parts
in
mutant
salts
frozen
twenty
exclusively
this
inorganic or
and
x g.
with
and
with
homogenizer
7.0)
found
144,000
out
harvested
pH
was
carried
AND BIOPHYSICAL
but
histamine,
oxalacetate
or
tyramine
pyruvate
activity. was
cultured
salt
(5
approximately
for
successive
many
generations
transfers 30%
100
as
much
of
in one
transaminase
drop
a medium into as
of 50 ml)
did
cells
the
7.
BIOCHEMICAL
Vol. 9, No. 2, 1962
AND BIOPHYSICAL
TABLE
Enzyme
Used
Pyridoxal
Phosphate
Requirement
Pyridoxal
Phosphate
AOD protein
at
435mp per mg per 10 minutes
t
0 0
Mutant crude supernatent
t
. 094 .064
Mutant fraction
t
.283 0
grown
on
putrescine.
mixture contained 0.1 ml of crude supernatant or partially purified enzyme, 15 pmoles of putrescine, 200 pmoles of Tris buffer pH9.0, 10 p.moles of oThe mutant 20 yof pyridoxal phosphate when used. protamine sulfate and ammonium sulfate treatment. later.
It thus
appears
that
the
transaminase
is
constitutive
in
mutant. The
--E.
coli
1959)
subsequent is
and
the
catabolism
same
consists
as
of
found
of the
in
succinic
The
first
reaction
the
DPN’ was
reaction.
were
incubated
obtained
supernatant
after
‘7
second
by
Thus, with a lag
when the
of
no
t
reduction
of
demonstrated
Fredericks,
six glutamate of
101
DPN’.
t glutamate
DPN+ by
as
by
the well
y -amino-
formation as
by
of coupling
7 -aminobutyrate
supernatant,
or
and
DPNH
a-ketoglutarate
approximately
reduction
semialdehyde
a-ketoglutarate,
crude
7 -aminobutyrate gave
the
was and
of
t DPNH
succinic
--t succinate
reaction
strain
(Jakoby
-aminobutyrate
*
demonstrated
y-aminobutyrate
third
a-ketoglutarate, crude
was The
from
t DPN+
this
reactions:
a-ketoglutarate
semialdehyde
butyraldehyde. glutamate
t
in
fluorescens
three
t DPN’
y -aminobutyrate
y -aminobutyraldehyde
Pseudomonas
following
y-aminobutyraldehyde
to
I
E. coli B crude supernatant
The 1 ml of reaction corresponding amount of 4pmoles of a-ketoglutarate, aminobenzaldehyde, and fraction was obtained after Details will be reported
this
RESEARCH COMMUNICATIONS
a rapid minutes. alone
and
reduction Incubation
with
DPN’
of
DPN’
of and
the
BIOCHEMICAL
Vol. 9, No. 2, 1962
The three
conversion
reactions
putrescine and
of putrescine
listed
Experimentally,
above
a DPN+
to
should
and
to T-labeled
C)terell,
AND BIOPHYSICAL
1947) was
y -aminobutyraldehyde lead
readily
followed
to the formation
a-ketoglutarate
succinate
RESEARCH COMMUNICATIONS
(identified
of succinate.
dependent
conversion
by paper
chromatography,
demonstrated
using
by the
dialyzed
of T-labeled Lugg
crude
supernatant.
Summary
A mutant
of _E. --coli B was obtained which can grow on putrescine C and N source. This mutant converts putrescine to succinate
sole
y -aminobutyraldehyde, manner was
y -aminobutyrate
as found
in Pseudomonas
not catalyzed
constitutive
strain
oxidase,
of --E.
via
semialdehyde
The first
fluorescens.
by a diamine
in this
and succinic
as the
in similar
reaction,
but by a transaminase
however, which
is
coli.
Acknowledgment The
work
was
supported
by U. S.P.
H. S. grants
(H-4139)
and (A-5384).
References 1.
Consden,
R.,
2.
Holmstedt,
3.
Jakoby,
4.
Lugg,
J.W.H.,
5.
Mora,
P. T.,
Young,
6.
Slotta,
K.H.,
and Primosigh,
7.
Tabor,
H.,
W.
Gordon,
A.H.,
B.,
Lasson,
L.,
B.,
and Fredericks, and Overell,
Tabor,
B.G.,
C. W.,
and Martin, and Tham, J., B. T.,
Biochem.
R.,
Biochim.
J. Biol.
Chem.,
Nature,
and Rizivi, J.,
A. J.P.,
S.,
Nature,
and Rosenthal,
102
160,
Biophys. 234,
-38, Acta,
224 (1944). 48,
182 (1961).
2145 (1959).
87 (1947).
J. Bio. 168,
J.,
Chem.,
237,
157 (1962).
696 (1951).
S. M.,
Ann.
Rev.
Biochem.,
-30,
579 (1961)