J. Nutr.
ROLE
OF
TO
VITAMIN
B12 AND
24, 477-489,
1978
RELATED
MUTASE
METHANOL-UTILIZING
Shunsaku
Vitaminol.,
ENZYMES
METHYLMALONYL-CoA PRO
Sci.
IN
A
BACTERIUM,
TAMINOBA
CTER
UEFA, K azuyoshi
R USER
SATO, and Shoichi
SHIMIZU1
Department of Food Science and Technology, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 454, Japan (Received April 21, 1978)
Summary
A
required
methanol-utilizing
cobalt
replaced
by
medium.
The
ion
or
succinate presence
extracts
of
fairly
high
detected
P.
a similar
extracts
of
P.
energy
source
ruber
the
extracts
of
- CoA
to
succinyl-CoA
species
B12,
workers
(1-5).
However,
seems
still
bacteria for
the
vitamin
P. and
ruber,
produces
showed
that
(methyl-B12)
a
In
by
to
us,
considerable forms
succinyl-CoA,
methylmalonyl-CoA
role
is
on
methanol
B12
produced
of
which
synthesis preliminary
from
which present
mutase
might
play
been
found
has
been
studied
to
a sole
in
clarify
carbon
In
a previous
ruber
were
by
1 上 田俊 策 ,佐 藤 一 精,清 水 祥 一 477
the of
a possible
important
ruber role
role
energy
source
paper
(3),
we
chromatography
and
that
methyl-B12 and
homocys
adenosyl-B12-dependent
conversion P.
several
methylcobalamin
paper
that
produce
by
and
P.
analysis,
to
methanol-utilizing
B12.
described
an
and
propionyl
N5-methyltetrahydrofolate
cells
also
cell-free
carbon
have
significant
enzyme
the
cell only
Furthermore, from
B12
by
catalyzes in
sole
reactions
identified
and
a
the not but
The
activity.
vitamin
vitamin
report
was
of
as
were
spectroscopy
be
to ƒ¿-hydroxyglutarate.
therefore
grows
organisms
as
the
was
period.
bacteria
them.
adenosyl-B12,
mutase,
these
by
vitamin
other
bacteria
amount
of
our
with cultivation
catalyzed
It
could
in
mutase
mutase
physiological
obscure.
in methionine addition,
methylmalonyl-CoA CoA
be
cobalt-deficient
the
this
a-ketoglutarate
in
to
compounds
methanol-utilizing
produced
absorption
participated
non-C1
from
which
demonstrated
of
methylmalonyl-CoA
the
isolated
the
the
productivity
to
B12
reported
on
was
activity
that
and
its
and
ultraviolet
teine.
and
specific
Tuber,
factor,
(adenosyl-B12)-dependent
mutase
microorganism
of
vitamin
Protaminobacter
growth
additions
throughout
grown had
this
(CoA)
with level
also
its
adenosylcobalamin
The
in comparison at
Several
ruber.
as
various
A
of
B12
among
methylmalonyl-coenzyme free
bacterium,
vitamin
of (6).
in
this
It
methylmalonyl
was
assumed
bacterium
that because
478
of
S. VEDA,
its
high
specific
shermanii
or
concerning
the
bypass
in
activity,
animal
the
as
tissues
K.
SATO,
compared
(7).
In
tricarboxylic
cycle,
Materials. were
droxyglutarate
(zinc
from
salt)
was
was
synthesized
methylmalonyl-CoA
was
prepared
as
paper
the
unlabeled
No.
50
developing SHEMIN tion
with
(9),
(10).
The
measured method
of
the
LIPMANN
The For
the
and
the
studies
glassware
was
deficient
medium,
et
al.
was
incubated
(12).
100-ml
purified
by
distillation.
follows:
Twenty
evaporator,
and
washed
the
This
treatment
residue
thus
obtained
FeCl3
was
Among (cyano-B12),
washed
with
residue
was
repeated
by
compounds and
HCl,
HCl
was three
measuring be
sodium P-cellulose
1M potassium
in
times in
the
to
20ml
glutamate packed phosphate
a small
at
to were in
the
purified
buffer
(pH
and
HCl
was
50W-X12 by
and
of
Fe3+
a rotary deionized
completely. water.
The
as
11cm).
that on
volume
The content
343nm.
medium,
a column
times
dryness
deionized
absorbance
supplemented
and
three
(1.6 ion
to
remove of
NaOH,
Methanol
Dowex
cobalt
of trace
Na2Mo04
water.
evaporated
dissolved
0.5%
a column
to remove
was
that
and
extracted
of
to
cobalt
procedure
ZnSO4,
a column applied
eluate
dissolved
to
follows:
1N
in
the
except
FeCl3
was
from All
The
modified
deionized
using
ion
medium
methanol,
mixture
from
were of
The
was finally
as 0.5N
HCl.
resulting
methionine
chromatography
purified
400ml
3N
a
H3BO3,
twice
this (6).
cobalt
water.
basal
to
chloroform.
was
remove
1-nitroso-2-naphthol the
throughout
deionization.
the
the
grade.
previously
deionized as
except
cooling,
(10mg/ml)
with of
determined the
0.12%
redistilled
FeCl3
with
were to
analytical
used
mixed-bed
same
medium
of
was
to
extrac
according
described
according
recrystallization FeCl3
of
50ml
water.
of
of
the
After
by
the
purified
us
ether
and
propionyl-CoA
were
the
SIMON
by
540nm
essential
washed was
of
by been
same
as
by
removed
materials
the Too filter
volume)
described
and
has
14C-Labeled by on
by
as
isolated
Unlabeled
(8).
chromatography
at
other
DL-ƒ¿-Hy Co.
acid
was
purified
and
of
20ml
by
ml
was
liter
30min.
portions purified
with
HCl
was
One
for
were
eluted
3N
the
discussed.
[U-14C]ƒ¿-keto
FLAVIN
(5:2:3,
it was
composition
with
45•Ž
Cu5O4
column
deficiency
omitted,
mixed
at
was
in
of
by
medium
was
and
Chemical
method
formed
ruber
basal
water
whose was
KLIEWER
The
cobalt
soaked
CoSO4•E7H2O
with
of
Glass-distilled
Sigma
prepared
All
P.
the
is also
Association.
hydroxamates
(11).
medium. of
system,
methylmalonyl-CoA their
TUTTLE
composition
medium.
elements
of
results
Furthermore,
[14C]Na2CO3
purified
acid
of
and
mutase
acid-water
propionic
detail.
METHODS
the
was
absorbance
Microorganism study.
free
the
experimental
2-[14C]methyl-malonic
and
concentrations
by
by
n-butanol-acetic
the
more
from
Propionyl-CoA
and
in
Propionibacterium
the
Radioisotope
from
compound
solvent.
system
Japan
obtained
methylmalonyl-CoA
method
with
acid,
purchased
with
deal
AND
2-[14C]Methyl-malonic
acid
reported
we
including
MATERIALS
glutaric
that
paper,
mutase
acid
S. SHIMIZU
with
this
methylmalonyl-CoA
and
by (0.6 7.5)
cyanocobalamin P-cellulose by and
3cm) purified
column was
pre water.
V. B12 AND
Each
sample
Sodium
was
- naphthol
from
5ml
cobalt-deficient
was
24,50
(ADS
For
culture
50hr,
(see
stoppers cotton.
order
was
followed
660nm.
The
relationship
1.
cells
300
to
The
wet
cells When
(pH
7.3)
for
(1g) they
supernatant
dialyzed
overnight
gentle
stirring
the
water.
1-nitroso-2
to
the
used after
and
centrifuged
cells
and
Fuji
obtained 5ml
used
optical
in
with at
In
this
place
cobalt
of
ion
30•Ž,
and
in the
spectrophotometer
OD
the
thus
as indicated.
medium
is
in
Fig,
1.
(O. D.).
500-ml middle
at
shown
density
in
at
cultivation
containing
were
and
grown
10 ml the by
2 liters
of
were
(3g) of
for
0.05M
grinding
Sakaguchi flasks logarithmic
0.01M
10min
phase
preparation. with
0.5g
13,000•~g
for
20min.
for
To
the
more
20min
buffer
(pH 10ml
for
and buffer
several
for
necessary,
charcoal
4•Ž.
a mortar
phosphate
of
0 to
phosphate
13,000•~g
When
treated
at
using
continued at
potassium
performed
potassium
was
centrifugation
enzyme was
operations
alumina
and
at
of ADS
All
with
a crude
ADS
containing of
stated.
obtained
as
tube
shaker
D. S.
of
were
disrupted,
grinding
Akiyama
harvested
extracts.
paste,
test
Japan) the
tubes
period
compounds Co.,
conditions,
test
The
a reciprocating
the value
and
to
each
of
value
sources
ground
against
was
an
otherwise
well
various
on
the
medium,
were
added
dialysate
with
times
to
Chem.
out
enzyme
were
The
homogenate
carried
cell free
was
cell
deionized
by
cobalt-deficient
contamination
between
unless
of
minutes.
This
5ml
479
cobalt-deficient
three
of
with
between
basal
350),
drop
inoculated
prevent
directly
used
Preparation
pestle.
to
Correlation
100ml
(ADS,
with
inoculation.
was
(Shinetsu
was
growth
The
P. RUBER
purified
under
least
One
supplemented
Cultivation
containing
IN
eluted
studies at
the final
50)
stoppers
in
Fig.
and
were
growth
before
about
medium "silico"
cotton
column
transferred
respectively.
below);
cobalt-deficient experiment,
the
were medium
and
the
SYSTEMS
procedure.
stock
value
to
ENZYME
deoxyribonucleosides
conditions.
cells
RELATED
applied
and
extraction Culture
the
then
succinate
ITS
30min
supernatant,
was 7.3). of
the
with solid
480
S. UEDA,
ammonium
sulfate
30min
and
0.05M
was
potassium
protein
was
albumin Enzyme
of
mixture
0.85ml.
The
15,000•~g
for
the
added
assayed
by
tris
to
the
the
the
method
total
basis
steps
for
protein),
50min
in
addition
(pH and
37•Ž
added
0.2ml
by
centrifugation
0.1ml
then
boiled
the
supernatant
lamp.
of for
of
a total
et
2N
at
supernatant
the
14min
and
the
of
method
of
placed
(10).
of
reaction
bring
total
supernatant to
succinic
acids
with were solution
2.5-ml evaporated were
a
pH
of to
subjected
of
al.
(17).
enzyme
by
The
boiling
0.03ml 3N
ether
to
was
the
(10ƒÊ
extracts at
30•Ž
terminated
the
by
precipitate
was
supernatant The
A
were
mixture
0.2-ml
was
aliquot
under
an
Tri-Carb
products.
The
of
of
infrared liquid
by
at
To
1N
the
acids
in the
vigorous redissolved chromatography
to
of
mixture
were
agitation.
The 0.1ml
(Togo filter
dark,
was
and
added
to
40min,
esters
the
and
then
six
times
methyl-malonic then
extracted
combined of
100ƒÊ
in a total
for thio
the
6.5),
the
water
15,000•~g
authentic
mixture
(pH
in
by 2-[14C]
protein),
10min
hydrolyze
stage,
in
for
at
KOH
this
buffer
(0.8mg
30•Ž
enzymatic
mainly
contained
maleate extracts
4min.
At
examined
mixture
centrifugation
HCl.
and paper
crude
incubated
Packard
was
reaction
crude
for
After
Organic
dryness
1ƒÊmole
dried a
. were
100ƒÊmoles;
also
15min.
reaction
(0.14ƒÊCi/ƒÊmole);
1.5ml. with
in
methylmalonyl-CoA
incubated
3 with
(11)
contained
7.5),
3min,
completely
for
3320-515).
was
to
for
at
assayed
mixture
of
the
compound
was
0.5ml
unless To
method
permanganate.
measured
mixture
added.
portions
and
the
treated
were
was
To
of 4%
vial
and
terminated
2 to
glass
1hr,
succinyl-CoA
(pH
for
1,000•~g
2 nmoles;
volume
acid
reaction
boiling
a total
3min.
were
reaction
the
a
(16).
centrifugation
2 nmoles;
40min.
at
vial
from
The
was
acidified
1.0ml
1ƒÊmole
was
the
for
et
After
mixture
and
After
and
analysis
CARDINALS
dark,
acid
a
succinyl-CoA
0.85ml.
the
in
for
[14C]Na2CO3,
The
al.
100ƒÊmoles;
for
to
The
to
et
permanganate-stable
1.1ml.
acid.
(model
adenosyl-B12,
volume
in
in in
methylmalonyl-CoA, moles;
bovine
enzyme,
dark
hydroxamic
adenosyl-B12, of
centrifuged
chromatographic
formation
using
6.5),
propionyl-CoA
al.
9,000•~g
perchloric
spectrometer
Paper
in The
according
the
3.0ml.
2ƒÊmoles;
volume
perchloric
radioactivity
scintillation
for
above.
STADTMAN
the
buffer
tube
2N
was
The
dissolved
(14),
boiling
of
of
FLAVIN
in
by to
of
glutathione,
stoppered
of
removed
stirring
was
assayed
volume
from
0.7ƒÊmole;
in
a
al.
and
buffer
at
radioactivity
of
was (15)
terminated
aliquots on
propionyl-CoA,
of
After
described
et
2 nmoles;
incubated
the
the
6ƒÊmoles;
Ci/ƒÊmole); (4.5mg
as
LOWRY
(hydroxymethyl)-aminomethane-hydrochloride
MgCl2•E6H2O,
the
of
STADTMAN
was
adjust
catalyzing
by
(13).
precipitate
dialyzed
maleate
2.0-ml
measuring
(succinate)
the
mutase by
was
content
enzymes
method
reaction
30min,
methylmalonyl-CoA The
saturation
and
adenosyl-B12,
mixture
and
was
the
contained 0.8ƒÊmole;
water
90%
7.3)
by
S. SHIMIZU
20min,
(pH
reported
mixture
stated,
to
for
buffer
method
methylmalonyl-CoA,
otherwise
up
Methylmalonyl-CoA
the
reaction
and
a standard.
assays. of
volume
slowly 13,000•~g
determined
as
modification The
at
phosphate
content
serum
added
centrifuging
K. SATO,
water.
ether Aliquots
paper
No.
and
extracts of 50,
this 2
by
V. B12 AND
40cm)
with
formic
the
completion
of
acids
on
green
intervals.
Each
in
piece
For
reduced 7.8),
of
1.0ml.
The
was for
treatments
used raphy
ether
by
was
vial
cut
its
481
saturated
paper
was
spraying
then
and
with dried
with
the
neutral
pieces was
10M
and
0.2%
into fifty
radioactivity
reaction
and
at
0.5-cm
measured
in
in
crude
incubated
the
supernatant
of
were
a
0.5ml
the
of
and
that
ether-benzene-formic
(5mg
30•Ž
1N
for
HCl.
were
same
as
[U-14C]ƒ¿dithiothre
0.2ƒÊmole;
extracts at
succinic, ƒ¿-ketoglutaric
standards
contained 0.5ƒÊmole;
dinucleotide,
the
was
addition
acids
mixture
MgCl2•E6H2O,
adenine
extraction
that
internal was
alcohol
by
the
mixture
organic
except
as
paper
a glass
30ƒÊmoles;
terminated 1hr,
after
formation,
isoamyl
located
nicotinamide
(pH
reaction
13,000•~g
The into
IN P. RUBER
the
(0.6ƒÊCi/ƒÊmole);
buffer
volume
the
n-butanol.
0.5ƒÊmole
25ƒÊmoles;
total
was
of ƒ¿-hydroxyglutarate,
acid,
phosphate
of
chromatography,
paper
put
SYSTEMS
spectrometer.
formation
ketoglutaric
ENZYME
consisting of
the
was
scintillation
itol,
solvent
After
bromocresol
RELATED
developing
acid.
position
liquid
ITS
1.5hr
protein), under
After
with
developing
described
acid-water
solvent
in
by
at
the
The
succinate acids
for
(21:9:7:2,
a
and
ether.
and ƒ¿-hydroxyglutaric
the
in N2,
centrifugation
extracted
those
potassium
of
paper
were
chromatog
volume).
RESULTS
Effect
of The
expected, 2,
P.
cobalt
since ruber
of
of
added. cobalt
The
When
the
P.
very
growth
little
was
growth
was
ruber of
of was
observed,
Effect
on
ion
the
or
growth
under
P.
B12
amount
ruber
for of
a strictly
observed
optimal
of
vitamin
a considerable
grow
the
B12
cobalt
it produced
0.1
of to
1.0ppm
inhibitory
to
cyano-B12
of The
various
for
additions
tested
stimulated
in
glutamate
and
grown the
at
1ppm
in
the
of
the
growth
vitamin
of
B12.
As
cobalt-deficient
in proportion
of
to cobalt
the ion
to
succinate proportion
or
could the
Optimal
The
P.
ruber
shown
was in
condition,
Fig.
and
concentration and
of
inhibited
at
a
cobalt 10ppm
effect
to
adequately
a to
in proportion
promoted was of
slightly
cobalt
ion
medium
Figure
on
with
lag growth
of
P.
ruber.
the
growth
of
P.
ruber
that
among
cobalt
ion.
succinate no
was
the
4
or
10
period
compounds
shows
the
above
any
of little
observed
longer on
various
cyano-B12
whether
replace
concentration had
with
also growth
determine B12.
supplemented
concentration
cobalt-deficient
vitamin
to
tested.
the made
deoxyribonucleosides range
3).
was
Although
a similar
was ion
(Fig.
growth.
medium growth
cyano-B12.
the
additions
cobalt
bacterial
cyano-B12
exerted
investigation
substitute
concentration
was
cyano-B12,
concentration
addition ppm
vitamin
ion.
concentrations to
or
of
could
stimulation ion
ion
requirement
effect
The
added. on
could the
several
growth
was
Methionine, growth
in
the
482
Fig.
S. USDA,
2.
Effect
added
at
ppb; •¡,
Fig.
3.
of cobalt the
0.1ppb; •¤,
Effect
added
following
at
, 1ppm
of cyano-B12 the
following
ion
on
the
K. SATO, and S. SHIMIZU
growth
of P. ruber.
concentrations: •¢, 0.01
ppb; •~,
on
the
0.001
growth
ppb
of
concentrations: •œ,
of CoSO4•E7H2O; •~,
no
To
the
10ppm; •›,
P. 1ppm
addition.
or
no
ruber. or
purified
medium,
1ppm; •£,
CoSO4•E7H20
100ppb; •œ
was
, 10ppb; •¥,
1
addition.
To
the
100ppb; •¥,
purified 10ppm
medium, or
cyano-B12 10ppb; •¢,
was 1ppb; •›
V. B12 AND
Fig.
4.
Effect
described 40mM 100ƒÊM
of in
sodium
various the
RELATED
additions
text
and
to
10mM 820ƒÊM
no
on
added
succinate; •¢,
methionine; •¡,
CoSO4•E7H2O; •~,
ITS
ENZYME
the the
growth purified
sodium
sodium
SYSTEMS
of
P.
ruber.
medium
succinate; •£,
glutamate
or
IN
at
Each the
2mM 400ƒÊM
P . RUBER
addition
following sodium
483
was
purified
as
concentrations: •œ, succinate; • ,
deoxyribonucleosides;
2, 0,
20,
or
3.6ƒÊM
addition.
Demonstration of methylmalonyl-CoA mutase in P. ruber From the results described above, we presumed the presence of methylmalonyl - CoA mutase in P. ruber. Table 1 shows that the disappearance of methylamonyl - CoA is dependent on the presence of adenosyl-B12. Methylmalonyl-CoA used in this experiment was a compound synthesized chemically and a racemic mixture. From this viewpoint, methylmalonyl-CoA was converted almost quantitatively to succinyl-CoA during the period of incubation. In the absence of adenosyl-B12, the amount of methylmalonyl-CoA which disappeared was markedly decreased. Table
1.
Requirement
of adenosyl-B12
for methylmalonyl-CoA
mutase
in P . ruber.
a 0 to 90% ammonium sulfate fraction (3mg of protein) prepared as describ ed in the text was used as an enzyme preparation. b Hydroxocobalamin .
484
S. VEDA,
However,
a little enzyme
K.
activity
SATO,
and
S. SHIMIZU
was detected,
presumably
owing
to the presence
of
endogenous adenosyl-B12. Cyano-B12, hydroxo-B12 and methyl-B12 could not substitute for adenosyl-B12 and seemed rather to inhibit this reaction. To confirm further the presence of methylmalonyl-CoA mutase, the enzymatic reaction
products
formed
from
14C-labeled
methylmalonyl-CoA
were examined
by
paper chromatography. The chromatogram shown in Fig. 5 indicates that the radioactive compound is clearly detected at the same position as the authentic succinic acid. Furthermore, a small amount of another radioactive compound appeared behind the solvent front, and its Rf value coincided acid, although not shown in the figure.
Fig. Fig.
5.
Paper
Succ,
5.
Fig.
chromatogram
succinic
acid;
with that
of reaction MM,
products
methylmalonic
acid.
derived For
from detail
6.
14C-labeled of
of fumaric
methylmalonyl-CoA.
experimental
procedures,
see
the
text. Fig.
6.
Time
course
conditions protein
of
of enzyme was
used
and
the
growth
reaction the
period
(•œ) were
and
the
as described of incubation
activity in was
of the
methylmalonyl-CoA
text
6min,
except according
that
mutase
(a)
approximately to
reference
0.3mg
. The of
(6).
Relationship of methylmalonyl-CoA mutase activity to the growth phase or to the growth substrates Figure 6 shows the growth of P. ruber and the specific activity of methylamonyl-CoA mutase. Although its specific activity was slightly lower at the earlier logarithmic growth phase, it remained approximately constant in the range of 110 to 140nmoles/min/mg of protein during the period of cultivation. On the other hand, P. ruber is a facultative methylotroph, which can grown even on non-C1 compounds as a sole carbon and energy source, and produce vitamin B12.When P.
V. B12 AND
Table
2.
ITS
Specific
RELATED
activity grown
ENZYME
SYSTEMS
of methylmalonyl-CoA on various
carbon
IN P . RUBER
mutase
485
in P. ruber
sources.a
a Cells were grown on each compound as a sole carbon and energy source and harvested at the middle logarithmic phase. b The conditions of enzyme reaction were as described in the text except that 0.3 to 0.4mg of protein was used and the period of incubation was 5min, according to reference (6).
ruber was grown on the substrates as indicated, the activity of the mutase was detected in all cases (Table 2). Incorporation of 14CO2into succinate For a better understanding of the role of methylmalonyl-CoA mutase in P. ruber, the precursor of methylmalonyl-CoA was investigated. Table 3 shows that the cell-free extracts of P. ruber catalyzed the 14CO2-fixation reaction with propionyl - CoA as a reactant to produce succinyl-CoA. In the absence of adenosyl-B12 or propionyl-CoA, the incorporation of 14CO2 was reduced, although a little radioactivity was observed owing to the endogenous compound. Even in the presence of biotin, the radioactivity did not increase dramatically beyond its level detected in the complete reaction system. Table
Reaction The by
paper
3.
products
formed
reaction
products
chromatography.
detected
in
mixture
incubated
experimental product.
Incorporation
the
into
derived
from
shown
90min. employed.
by cell-free
by
extracts
cell free
of P.
extracts
14C-labeled ƒ¿-ketoglutarate in
corresponding for
succinate
from ƒ¿-ketoglutarate
As
position
conditions
of 14CO2
Fig. to
However, Succinate
7, the
ruber.
of
P.
radioactive
activity was
was also
examined
compound
could
authentic ƒ¿-hydroxyglutaric its
ruber
were
acid not
confirmed
high as
in
under a
reaction
be the the
486
Fig.
S. VEDA,
7. -HG
Paper
chromatogram
,ƒ¿-hydroxyglutaric
of
K. SATO,
reaction
products
acid;ƒ¿-KG,ƒ¿-ketoglutaric
and
S. SHIMIZU
derived acid;
from Succ,
14C-labeled ƒ¿-ketoglutarate.ƒ¿ succinic
acid.
For
detail
of
experimental procedures,seethe text. DISCUSSION In the previous paper (3), we showed that a methanol-utilizing bacterium, P. ruber, could produce a large amount of vitamin B12 in the two different forms, i.e., methyl-B12 and adenosyl-B12. The results concerning the effect of cobalt ion or cyano-B12 on the growth of P. ruber indicate that these two B12 compounds are essential for the growth of this bacterium. When cyano-B12, which was used in this study because of its stability, was added to the cobalt-deficient medium, a slightly longer lag period was observed. This phenomenon might partly be due to some difficulty in the incorporation of cyano-B12 into the cells and partly due to that in the transformation from cyano-B12 into the active forms such as methyl-B12 and adenosyl-B12. In this regard, TAMAOet al. (18) reported that the transformation from cyano-B12 into adenosyl-B12 occurred with some difficulty in the cells of P. shermanii. As mentioned above, P. ruber produces vitamin B12 in the forms of methyl-B12 and adenosyl-B12. This implies that there are at least two different vitamin B12 dependent enzyme systems in the cells of P. ruber. We have already demonstrated that methyl-B12 participates in methionine synthesis from homocysteine and N5 - methyltetrahydrofolate in the cells of P. ruber (3). In spite of the occurrence of B12 - dependent methionine synthetase, methionine had little effect on the growth, so far as examined. Although it is difficult to provide a definite explanation at the present stage of this study, one can speculate that if the absolute amount of cobalt, which is converted into vitamin B12 and required for methionine synthesis in P. ruber, is
V. B12 AND
ITS
RELATED
ENZYME
SYSTEMS
IN P. RUBER
487
small, this bacterium would be able to synthesize methionine with a trace amount of cobalt ion remaining in the inoculum or in the purified medium. This interpretation might be supported by the result that the cells grown on succinate under cobalt deficient conditions had methionine synthetase activity corresponding to about 10% of that in the cells grown under cobalt-sufficient conditions (unpublished data). Further investigation is needed for any final conclusion. On the other hand, adenosyl-B12 is well known to function as the coenzyme of methylmalonyl-CoA mutase in animal tissues and microorganisms such as P. shermanii. As the addition of succinate to the cobalt-deficient medium resulted in the sufficient stimulation of growth, we presumed the existence of methylmalonyl-CoA mutase in the cells of P. ruber. This presumption could be demonstrated from the results that the disappearance of methylmalonyl-CoA used as a reactant was dependent on the presence of adenosyl-B12 and that succinyl-CoA was detected as an enzymatic reaction product in the 14C-labeling experiment. This finding is of interest because of the first observation from methanol-utilizing bacteria. Additionally, this mutase system seems to play an important role in the metabolism of P. ruber because of its fairly specific activity. In this connection BARKER (7) reported that the specific activity of the mutase was in the range of 10 to 100 nmoles/min/mg of protein in P. shermanii, which is known as one of the best producers of this mutase. The mutase activity could be detected in the cells of P. ruber, even when grown on non-C1 compounds as a sole carbon and energy source (Table 2). Furthermore, an almost constant level of its specific activity was found irrespective of the bacterial growth phase. This observation is completely consistent with our previous result as to the variation in the forms of B12 compounds during growth; adenosyl-B12 was found throughout the cultivation period (3). On the basis of these facts, at least part of the adenosyl-B12 formed functions as a coenzyme of methylmalonyl-CoA mutase in P. ruber, and this enzyme system seems to play a fundamental role in the metabolism of this bacterium. However, there may be other adenosyl-B12 - dependent enzyme system(s) apart from methylmalonyl-CoA mutase in P. ruber because of the incomplete effect of succinate on growth.
Fig.
8. in
Proposed Rhodopseudomonas
metabolic
sequence spheroides.
leading
to
the
formation
of succinate
via ƒ¿-hydroxyglutarate
488
S. VEDA,
In was
recent
years,
proposed
the
with
(19).
In
It
was
participated the
to
which
is
results,
the
reverse
we
the
the
of of
course
of
ruber
presence
of
the
the
study
via
this
catalyzed
of
route.
the
propionyl-CoA
this
the
from
8,
present
propionyl
carboxylase
in
P.
ruber.
from ƒ¿-ketoglutarate, dehydrogenase
of
Fig.
glutamate
In
steps
of ƒ¿-hydroxyglutarate
possibility
in
bacterium,
methylmalonyl-CoA
of ƒ¿-hydroxyglutarate
on
illustrated
B12-producing
succinate
P.
formation
reaction
speculated
cycle,
vitamin
adenosyl-B12-dependent
formation
the
observed
acid
and
extracts
implying
we
S. SHIMIZU
tricarboxylic
that
the
cell-free
succinate,
addition,
the
and
during
indicated in
investigation, CoA
of
SATO,
photosynthetic
spheroides,
metabolism mutase
bypass
the
Rhodopseudomonas
K.
bypass
(20).
in
P.
From
these
ruber.
REFERENCES
1)
2)
NISHIO,
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vitamin
B12
YANG,
production.
T.,
NISHIO,
N.,
YANG,
SATO,
K.,
utilizing 4)
and
VEDA,
S.,
A.,
TORAYA,
T.,
SATO,
K.,
Y.,
YONGSMITH,
VEDA,
9)
BARKER,
H.
10)
A.
M.
Kaplan,
N. O.,
SIMON,
E. 75,
FLAVIN,
J.,
and
13)
M.,
and
F.,
KLIEWER,
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16)
17)
OCHOA,
and
M.,
Folin
STADTMAN,
TUTTLE,
vitamin
B12
production
39,
207-213.
its
role
and 33,
in
by
a methanol
515-521.
Production
of
Vitamin
B12
vitamin
B12
by
921-924.
S.
(1975):
production
by
a
mutase
in
a methanol-utilizing
248-250. mutase,
Methods
in The
in
Vol.
6,
preparation
R.,
and
E.
R.
Colowick,
vitamin
coenzyme
B12
CARDINALE,
G.
Enzymes,
Enzymology, pp.
of
ed.
by
Boyer,
P.
D.,
ed.
by
Colowick,
S.,
and
538-539.
S-succinyl
coenzyme
A.
J. Am.
Chem.
Arch.
J.,
Biochem.
FARR,
J.
P.,
tissue
EVANS,
P. M.,
and
Biophys.,
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H.,
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(1964):
tissues.
the
A
I.
Enzymatic
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biological
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92-99.
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RANDALL,
of
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aryl
cobalt
R.
and
II.
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Protein
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Acad.
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Press LYNEN,
ABELES, level
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Biochem.
B12-coenzyme 131,
193,
Acadmic
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EGGERER,
vitamin
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Methylmalonyl
Chem.,
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propionate
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Proc. A.
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Kaplan,
OVERATH,
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229,
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Preparation
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Chem.,
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and
of propionic
specific
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21,
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P. A., Soil,
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from
J., DREYFUS,
its effect
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reagent.
S. P., R.,
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WOOD,
(1957):
E.
C.
Plant
ROSEBROUGH,
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159,
MAYEUX,
enzyme
phenol
Metabolism
succinate. L.
LOWE,
STADTMAN,
CoA.
its
477-479.
71,
York,
The
and
509-537. in
New
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to
Chem.,
the
H.,
the
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6, pp.
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J. Biol.
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LOWRY, O.
by
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ed.
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with
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Methylmalonyl-CoA,
of propionate
properties 14)
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FUKUI,
B12-dependent
York,
B12
Methylmalonyl-CoA
FEBS
Coenzyme New
Chem.,
Microbiol.,
52,
30,
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bacteria
2520.
LIPMANN,
using
Inc.,
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of
Biol.
vitamin
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Technol.,
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of Environ.
FUKUI,
A.,
methanol-utilizing
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Agric.
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101.
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Protaminobacter
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Further
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of
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sp.
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Isolation 39,
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bacterium.
bacterium, 7)
SHIMIZU,
bacteria.
methanol-utilizing 6)
T.
Klebsiella
and
OHYA,
(1975):
Chem.,
Protaminobacter
methanol-assimilating 5)
T.
Biol.
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bacterium,
TANAKA,
KAMIKUBO, Agric.
T.,
methanol-utilizing 3)
and
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Biophys. H. on
York,
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The Res.
in Enzymology,
Vol. role
Commun.,
Experimental
metabolism
3, pp. of
228-231. biotin
2,
and
1-7.
vitamin
of methylmalonyl
B12
V. B12 AND
18)
TAMAO, related in
Y.,
19) OHMORI,
28, H.,
purple
20) OKUYAMA, glyoxylic 110,
T.,
RELATED
SHMIZU,
XVI.
S.,
and
Comparison
ENZYME
FUKUI,
66-80.
S. (1963):
coenzyme
IN
P. RUBER
Chemical
studies
of cyanocobalamin formation
in and
by
489
vitamin
B12
and
its
hydroxocobalamin
Propionibacterium
shermanii
.
578-582. ISHITANI,
nonsulfur M., acid
SYSTEMS
of availabilities
5,6-dimethylbenzimidazolylcobamide
Vitamins,
in
KATO,
compounds.
ITS
H.,
SATO,
bacteria: TSUIKI,
catalyzed
S., by
K.,
SHIMIZU,
Participation and enzymes
KIKUCHI, from
S., of G.
and
vitamin
FUKUI, B12.
S. (1974):
Metabolism
Agric.
Chem.,
Biol.
(1965): ƒ¿-Ketoglutarate-dependent
Rhodopseudomonas
spheroides.
of glutamate 38,
359-365 oxidation
Biochim.
Biophys.
of Acta,
ROLE
OF
TO
VITAMIN
B12 AND
24, 477-489,
1978
RELATED
MUTASE
METHANOL-UTILIZING
Shunsaku
Vitaminol.,
ENZYMES
METHYLMALONYL-CoA PRO
Sci.
IN
A
BACTERIUM,
TAMINOBA
CTER
UEFA, K azuyoshi
R USER
SATO, and Shoichi
SHIMIZU1
Department of Food Science and Technology, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 454, Japan (Received April 21, 1978)
Summary
A
required
methanol-utilizing
cobalt
replaced
by
medium.
The
ion
or
succinate presence
extracts
of
fairly
high
detected
P.
a similar
extracts
of
P.
energy
source
ruber
the
extracts
of
- CoA
to
succinyl-CoA
species
B12,
workers
(1-5).
However,
seems
still
bacteria for
the
vitamin
P. and
ruber,
produces
showed
that
(methyl-B12)
a
In
by
to
us,
considerable forms
succinyl-CoA,
methylmalonyl-CoA
role
is
on
methanol
B12
produced
of
which
synthesis preliminary
from
which present
mutase
might
play
been
found
has
been
studied
to
a sole
in
clarify
carbon
In
a previous
ruber
were
by
1 上 田俊 策 ,佐 藤 一 精,清 水 祥 一 477
the of
a possible
important
ruber role
role
energy
source
paper
(3),
we
chromatography
and
that
methyl-B12 and
homocys
adenosyl-B12-dependent
conversion P.
several
methylcobalamin
paper
that
produce
by
and
P.
analysis,
to
methanol-utilizing
B12.
described
an
and
propionyl
N5-methyltetrahydrofolate
cells
also
cell-free
carbon
have
significant
enzyme
the
cell only
Furthermore, from
B12
by
catalyzes in
sole
reactions
identified
and
a
the not but
The
activity.
vitamin
vitamin
report
was
of
as
were
spectroscopy
be
to ƒ¿-hydroxyglutarate.
therefore
grows
organisms
as
the
was
period.
bacteria
them.
adenosyl-B12,
mutase,
these
by
vitamin
other
bacteria
amount
of
our
with cultivation
catalyzed
It
could
in
mutase
mutase
physiological
obscure.
in methionine addition,
methylmalonyl-CoA CoA
be
cobalt-deficient
the
this
a-ketoglutarate
in
to
compounds
methanol-utilizing
produced
absorption
participated
non-C1
from
which
demonstrated
of
methylmalonyl-CoA
the
isolated
the
the
productivity
to
B12
reported
on
was
activity
that
and
its
and
ultraviolet
teine.
and
specific
Tuber,
factor,
(adenosyl-B12)-dependent
mutase
microorganism
of
vitamin
Protaminobacter
growth
additions
throughout
grown had
this
(CoA)
with level
also
its
adenosylcobalamin
The
in comparison at
Several
ruber.
as
various
A
of
B12
among
methylmalonyl-coenzyme free
bacterium,
vitamin
of (6).
in
this
It
methylmalonyl
was
assumed
bacterium
that because
478
of
S. VEDA,
its
high
specific
shermanii
or
concerning
the
bypass
in
activity,
animal
the
as
tissues
K.
SATO,
compared
(7).
In
tricarboxylic
cycle,
Materials. were
droxyglutarate
(zinc
from
salt)
was
was
synthesized
methylmalonyl-CoA
was
prepared
as
paper
the
unlabeled
No.
50
developing SHEMIN tion
with
(9),
(10).
The
measured method
of
the
LIPMANN
The For
the
and
the
studies
glassware
was
deficient
medium,
et
al.
was
incubated
(12).
100-ml
purified
by
distillation.
follows:
Twenty
evaporator,
and
washed
the
This
treatment
residue
thus
obtained
FeCl3
was
Among (cyano-B12),
washed
with
residue
was
repeated
by
compounds and
HCl,
HCl
was three
measuring be
sodium P-cellulose
1M potassium
in
times in
the
to
20ml
glutamate packed phosphate
a small
at
to were in
the
purified
buffer
(pH
and
HCl
was
50W-X12 by
and
of
Fe3+
a rotary deionized
completely. water.
The
as
11cm).
that on
volume
The content
343nm.
medium,
a column
times
dryness
deionized
absorbance
supplemented
and
three
(1.6 ion
to
remove of
NaOH,
Methanol
Dowex
cobalt
of trace
Na2Mo04
water.
evaporated
dissolved
0.5%
a column
to remove
was
that
and
extracted
of
to
cobalt
procedure
ZnSO4,
a column applied
eluate
dissolved
to
follows:
1N
in
the
except
FeCl3
was
from All
The
modified
deionized
using
ion
medium
methanol,
mixture
from
were of
The
was finally
as 0.5N
HCl.
resulting
methionine
chromatography
purified
400ml
3N
a
H3BO3,
twice
this (6).
cobalt
water.
basal
to
chloroform.
was
remove
1-nitroso-2-naphthol the
throughout
deionization.
the
the
grade.
previously
deionized as
except
cooling,
(10mg/ml)
with of
determined the
0.12%
redistilled
FeCl3
with
were to
analytical
used
mixed-bed
same
medium
of
was
to
extrac
according
described
according
recrystallization FeCl3
of
50ml
water.
of
of
the
After
by
the
purified
us
ether
and
propionyl-CoA
were
the
SIMON
by
540nm
essential
washed was
of
by been
same
as
by
removed
materials
the Too filter
volume)
described
and
has
14C-Labeled by on
by
as
isolated
Unlabeled
(8).
chromatography
at
other
DL-ƒ¿-Hy Co.
acid
was
purified
and
of
20ml
by
ml
was
liter
30min.
portions purified
with
HCl
was
One
for
were
eluted
3N
the
discussed.
[U-14C]ƒ¿-keto
FLAVIN
(5:2:3,
it was
composition
with
45•Ž
Cu5O4
column
deficiency
omitted,
mixed
at
was
in
of
by
medium
was
and
Chemical
method
formed
ruber
basal
water
whose was
KLIEWER
The
cobalt
soaked
CoSO4•E7H2O
with
of
Glass-distilled
Sigma
prepared
All
P.
the
is also
Association.
hydroxamates
(11).
medium. of
system,
methylmalonyl-CoA their
TUTTLE
composition
medium.
elements
of
results
Furthermore,
[14C]Na2CO3
purified
acid
of
and
mutase
acid-water
propionic
detail.
METHODS
the
was
absorbance
Microorganism study.
free
the
experimental
2-[14C]methyl-malonic
and
concentrations
by
by
n-butanol-acetic
the
more
from
Propionyl-CoA
and
in
Propionibacterium
the
Radioisotope
from
compound
solvent.
system
Japan
obtained
methylmalonyl-CoA
method
with
acid,
purchased
with
deal
AND
2-[14C]Methyl-malonic
acid
reported
we
including
MATERIALS
glutaric
that
paper,
mutase
acid
S. SHIMIZU
with
this
methylmalonyl-CoA
and
by (0.6 7.5)
cyanocobalamin P-cellulose by and
3cm) purified
column was
pre water.
V. B12 AND
Each
sample
Sodium
was
- naphthol
from
5ml
cobalt-deficient
was
24,50
(ADS
For
culture
50hr,
(see
stoppers cotton.
order
was
followed
660nm.
The
relationship
1.
cells
300
to
The
wet
cells When
(pH
7.3)
for
(1g) they
supernatant
dialyzed
overnight
gentle
stirring
the
water.
1-nitroso-2
to
the
used after
and
centrifuged
cells
and
Fuji
obtained 5ml
used
optical
in
with at
In
this
place
cobalt
of
ion
30•Ž,
and
in the
spectrophotometer
OD
the
thus
as indicated.
medium
is
in
Fig,
1.
(O. D.).
500-ml middle
at
shown
density
in
at
cultivation
containing
were
and
grown
10 ml the by
2 liters
of
were
(3g) of
for
0.05M
grinding
Sakaguchi flasks logarithmic
0.01M
10min
phase
preparation. with
0.5g
13,000•~g
for
20min.
for
To
the
more
20min
buffer
(pH 10ml
for
and buffer
several
for
necessary,
charcoal
4•Ž.
a mortar
phosphate
of
0 to
phosphate
13,000•~g
When
treated
at
using
continued at
potassium
performed
potassium
was
centrifugation
enzyme was
operations
alumina
and
at
of ADS
All
with
a crude
ADS
containing of
stated.
obtained
as
tube
shaker
D. S.
of
were
disrupted,
grinding
Akiyama
harvested
extracts.
paste,
test
Japan) the
tubes
period
compounds Co.,
conditions,
test
The
a reciprocating
the value
and
to
each
of
value
sources
ground
against
was
an
otherwise
well
various
on
the
medium,
were
added
dialysate
with
times
to
Chem.
out
enzyme
were
The
homogenate
carried
cell free
was
cell
deionized
by
cobalt-deficient
contamination
between
unless
of
minutes.
This
5ml
479
cobalt-deficient
three
of
with
between
basal
350),
drop
inoculated
prevent
directly
used
Preparation
pestle.
to
Correlation
100ml
(ADS,
with
inoculation.
was
(Shinetsu
was
growth
The
P. RUBER
purified
under
least
One
supplemented
Cultivation
containing
IN
eluted
studies at
the final
50)
stoppers
in
Fig.
and
were
growth
before
about
medium "silico"
cotton
column
transferred
respectively.
below);
cobalt-deficient experiment,
the
were medium
and
the
SYSTEMS
procedure.
stock
value
to
ENZYME
deoxyribonucleosides
conditions.
cells
RELATED
applied
and
extraction Culture
the
then
succinate
ITS
30min
supernatant,
was 7.3). of
the
with solid
480
S. UEDA,
ammonium
sulfate
30min
and
0.05M
was
potassium
protein
was
albumin Enzyme
of
mixture
0.85ml.
The
15,000•~g
for
the
added
assayed
by
tris
to
the
the
the
method
total
basis
steps
for
protein),
50min
in
addition
(pH and
37•Ž
added
0.2ml
by
centrifugation
0.1ml
then
boiled
the
supernatant
lamp.
of for
of
a total
et
2N
at
supernatant
the
14min
and
the
of
method
of
placed
(10).
of
reaction
bring
total
supernatant to
succinic
acids
with were solution
2.5-ml evaporated were
a
pH
of to
subjected
of
al.
(17).
enzyme
by
The
boiling
0.03ml 3N
ether
to
was
the
(10ƒÊ
extracts at
30•Ž
terminated
the
by
precipitate
was
supernatant The
A
were
mixture
0.2-ml
was
aliquot
under
an
Tri-Carb
products.
The
of
of
infrared liquid
by
at
To
1N
the
acids
in the
vigorous redissolved chromatography
to
of
mixture
were
agitation.
The 0.1ml
(Togo filter
dark,
was
and
added
to
40min,
esters
the
and
then
six
times
methyl-malonic then
extracted
combined of
100ƒÊ
in a total
for thio
the
6.5),
the
water
15,000•~g
authentic
mixture
(pH
in
by 2-[14C]
protein),
10min
hydrolyze
stage,
in
for
at
KOH
this
buffer
(0.8mg
30•Ž
enzymatic
mainly
contained
maleate extracts
4min.
At
examined
mixture
centrifugation
HCl.
and paper
crude
incubated
Packard
was
reaction
crude
for
After
Organic
dryness
1ƒÊmole
dried a
. were
100ƒÊmoles;
also
15min.
reaction
(0.14ƒÊCi/ƒÊmole);
1.5ml. with
in
methylmalonyl-CoA
incubated
3 with
(11)
contained
7.5),
3min,
completely
for
3320-515).
was
to
for
at
assayed
mixture
of
the
compound
was
0.5ml
unless To
method
permanganate.
measured
mixture
added.
portions
and
the
treated
were
was
To
of 4%
vial
and
terminated
2 to
glass
1hr,
succinyl-CoA
(pH
for
1,000•~g
2 nmoles;
volume
acid
reaction
boiling
a total
3min.
were
reaction
the
a
(16).
centrifugation
2 nmoles;
40min.
at
vial
from
The
was
acidified
1.0ml
1ƒÊmole
was
the
for
et
After
mixture
and
After
and
analysis
CARDINALS
dark,
acid
a
succinyl-CoA
0.85ml.
the
in
for
[14C]Na2CO3,
The
al.
100ƒÊmoles;
for
to
The
to
et
permanganate-stable
1.1ml.
acid.
(model
adenosyl-B12,
volume
in
in in
methylmalonyl-CoA, moles;
bovine
enzyme,
dark
hydroxamic
adenosyl-B12, of
centrifuged
chromatographic
formation
using
6.5),
propionyl-CoA
al.
9,000•~g
perchloric
spectrometer
Paper
in The
according
the
3.0ml.
2ƒÊmoles;
volume
perchloric
radioactivity
scintillation
for
above.
STADTMAN
the
buffer
tube
2N
was
The
dissolved
(14),
boiling
of
of
FLAVIN
in
by to
of
glutathione,
stoppered
of
removed
stirring
was
assayed
volume
from
0.7ƒÊmole;
in
a
al.
and
buffer
at
radioactivity
of
was (15)
terminated
aliquots on
propionyl-CoA,
of
After
described
et
2 nmoles;
incubated
the
the
6ƒÊmoles;
Ci/ƒÊmole); (4.5mg
as
LOWRY
(hydroxymethyl)-aminomethane-hydrochloride
MgCl2•E6H2O,
the
of
STADTMAN
was
adjust
catalyzing
by
(13).
precipitate
dialyzed
maleate
2.0-ml
measuring
(succinate)
the
mutase by
was
content
enzymes
method
reaction
30min,
methylmalonyl-CoA The
saturation
and
adenosyl-B12,
mixture
and
was
the
contained 0.8ƒÊmole;
water
90%
7.3)
by
S. SHIMIZU
20min,
(pH
reported
mixture
stated,
to
for
buffer
method
methylmalonyl-CoA,
otherwise
up
Methylmalonyl-CoA
the
reaction
and
a standard.
assays. of
volume
slowly 13,000•~g
determined
as
modification The
at
phosphate
content
serum
added
centrifuging
K. SATO,
water.
ether Aliquots
paper
No.
and
extracts of 50,
this 2
by
V. B12 AND
40cm)
with
formic
the
completion
of
acids
on
green
intervals.
Each
in
piece
For
reduced 7.8),
of
1.0ml.
The
was for
treatments
used raphy
ether
by
was
vial
cut
its
481
saturated
paper
was
spraying
then
and
with dried
with
the
neutral
pieces was
10M
and
0.2%
into fifty
radioactivity
reaction
and
at
0.5-cm
measured
in
in
crude
incubated
the
supernatant
of
were
a
0.5ml
the
of
and
that
ether-benzene-formic
(5mg
30•Ž
1N
for
HCl.
were
same
as
[U-14C]ƒ¿dithiothre
0.2ƒÊmole;
extracts at
succinic, ƒ¿-ketoglutaric
standards
contained 0.5ƒÊmole;
dinucleotide,
the
was
addition
acids
mixture
MgCl2•E6H2O,
adenine
extraction
that
internal was
alcohol
by
the
mixture
organic
except
as
paper
a glass
30ƒÊmoles;
terminated 1hr,
after
formation,
isoamyl
located
nicotinamide
(pH
reaction
13,000•~g
The into
IN P. RUBER
the
(0.6ƒÊCi/ƒÊmole);
buffer
volume
the
n-butanol.
0.5ƒÊmole
25ƒÊmoles;
total
was
of ƒ¿-hydroxyglutarate,
acid,
phosphate
of
chromatography,
paper
put
SYSTEMS
spectrometer.
formation
ketoglutaric
ENZYME
consisting of
the
was
scintillation
itol,
solvent
After
bromocresol
RELATED
developing
acid.
position
liquid
ITS
1.5hr
protein), under
After
with
developing
described
acid-water
solvent
in
by
at
the
The
succinate acids
for
(21:9:7:2,
a
and
ether.
and ƒ¿-hydroxyglutaric
the
in N2,
centrifugation
extracted
those
potassium
of
paper
were
chromatog
volume).
RESULTS
Effect
of The
expected, 2,
P.
cobalt
since ruber
of
of
added. cobalt
The
When
the
P.
very
growth
little
was
growth
was
ruber of
of was
observed,
Effect
on
ion
the
or
growth
under
P.
B12
amount
ruber
for of
a strictly
observed
optimal
of
vitamin
a considerable
grow
the
B12
cobalt
it produced
0.1
of to
1.0ppm
inhibitory
to
cyano-B12
of The
various
for
additions
tested
stimulated
in
glutamate
and
grown the
at
1ppm
in
the
of
the
growth
vitamin
of
B12.
As
cobalt-deficient
in proportion
of
to cobalt
the ion
to
succinate proportion
or
could the
Optimal
The
P.
ruber
shown
was in
condition,
Fig.
and
concentration and
of
inhibited
at
a
cobalt 10ppm
effect
to
adequately
a to
in proportion
promoted was of
slightly
cobalt
ion
medium
Figure
on
with
lag growth
of
P.
ruber.
the
growth
of
P.
ruber
that
among
cobalt
ion.
succinate no
was
the
4
or
10
period
compounds
shows
the
above
any
of little
observed
longer on
various
cyano-B12
whether
replace
concentration had
with
also growth
determine B12.
supplemented
concentration
cobalt-deficient
vitamin
to
tested.
the made
deoxyribonucleosides range
3).
was
Although
a similar
was ion
(Fig.
growth.
medium growth
cyano-B12.
the
additions
cobalt
bacterial
cyano-B12
exerted
investigation
substitute
concentration
was
cyano-B12,
concentration
addition ppm
vitamin
ion.
concentrations to
or
of
could
stimulation ion
ion
requirement
effect
The
added. on
could the
several
growth
was
Methionine, growth
in
the
482
Fig.
S. USDA,
2.
Effect
added
at
ppb; •¡,
Fig.
3.
of cobalt the
0.1ppb; •¤,
Effect
added
following
at
, 1ppm
of cyano-B12 the
following
ion
on
the
K. SATO, and S. SHIMIZU
growth
of P. ruber.
concentrations: •¢, 0.01
ppb; •~,
on
the
0.001
growth
ppb
of
concentrations: •œ,
of CoSO4•E7H2O; •~,
no
To
the
10ppm; •›,
P. 1ppm
addition.
or
no
ruber. or
purified
medium,
1ppm; •£,
CoSO4•E7H20
100ppb; •œ
was
, 10ppb; •¥,
1
addition.
To
the
100ppb; •¥,
purified 10ppm
medium, or
cyano-B12 10ppb; •¢,
was 1ppb; •›
V. B12 AND
Fig.
4.
Effect
described 40mM 100ƒÊM
of in
sodium
various the
RELATED
additions
text
and
to
10mM 820ƒÊM
no
on
added
succinate; •¢,
methionine; •¡,
CoSO4•E7H2O; •~,
ITS
ENZYME
the the
growth purified
sodium
sodium
SYSTEMS
of
P.
ruber.
medium
succinate; •£,
glutamate
or
IN
at
Each the
2mM 400ƒÊM
P . RUBER
addition
following sodium
483
was
purified
as
concentrations: •œ, succinate; • ,
deoxyribonucleosides;
2, 0,
20,
or
3.6ƒÊM
addition.
Demonstration of methylmalonyl-CoA mutase in P. ruber From the results described above, we presumed the presence of methylmalonyl - CoA mutase in P. ruber. Table 1 shows that the disappearance of methylamonyl - CoA is dependent on the presence of adenosyl-B12. Methylmalonyl-CoA used in this experiment was a compound synthesized chemically and a racemic mixture. From this viewpoint, methylmalonyl-CoA was converted almost quantitatively to succinyl-CoA during the period of incubation. In the absence of adenosyl-B12, the amount of methylmalonyl-CoA which disappeared was markedly decreased. Table
1.
Requirement
of adenosyl-B12
for methylmalonyl-CoA
mutase
in P . ruber.
a 0 to 90% ammonium sulfate fraction (3mg of protein) prepared as describ ed in the text was used as an enzyme preparation. b Hydroxocobalamin .
484
S. VEDA,
However,
a little enzyme
K.
activity
SATO,
and
S. SHIMIZU
was detected,
presumably
owing
to the presence
of
endogenous adenosyl-B12. Cyano-B12, hydroxo-B12 and methyl-B12 could not substitute for adenosyl-B12 and seemed rather to inhibit this reaction. To confirm further the presence of methylmalonyl-CoA mutase, the enzymatic reaction
products
formed
from
14C-labeled
methylmalonyl-CoA
were examined
by
paper chromatography. The chromatogram shown in Fig. 5 indicates that the radioactive compound is clearly detected at the same position as the authentic succinic acid. Furthermore, a small amount of another radioactive compound appeared behind the solvent front, and its Rf value coincided acid, although not shown in the figure.
Fig. Fig.
5.
Paper
Succ,
5.
Fig.
chromatogram
succinic
acid;
with that
of reaction MM,
products
methylmalonic
acid.
derived For
from detail
6.
14C-labeled of
of fumaric
methylmalonyl-CoA.
experimental
procedures,
see
the
text. Fig.
6.
Time
course
conditions protein
of
of enzyme was
used
and
the
growth
reaction the
period
(•œ) were
and
the
as described of incubation
activity in was
of the
methylmalonyl-CoA
text
6min,
except according
that
mutase
(a)
approximately to
reference
0.3mg
. The of
(6).
Relationship of methylmalonyl-CoA mutase activity to the growth phase or to the growth substrates Figure 6 shows the growth of P. ruber and the specific activity of methylamonyl-CoA mutase. Although its specific activity was slightly lower at the earlier logarithmic growth phase, it remained approximately constant in the range of 110 to 140nmoles/min/mg of protein during the period of cultivation. On the other hand, P. ruber is a facultative methylotroph, which can grown even on non-C1 compounds as a sole carbon and energy source, and produce vitamin B12.When P.
V. B12 AND
Table
2.
ITS
Specific
RELATED
activity grown
ENZYME
SYSTEMS
of methylmalonyl-CoA on various
carbon
IN P . RUBER
mutase
485
in P. ruber
sources.a
a Cells were grown on each compound as a sole carbon and energy source and harvested at the middle logarithmic phase. b The conditions of enzyme reaction were as described in the text except that 0.3 to 0.4mg of protein was used and the period of incubation was 5min, according to reference (6).
ruber was grown on the substrates as indicated, the activity of the mutase was detected in all cases (Table 2). Incorporation of 14CO2into succinate For a better understanding of the role of methylmalonyl-CoA mutase in P. ruber, the precursor of methylmalonyl-CoA was investigated. Table 3 shows that the cell-free extracts of P. ruber catalyzed the 14CO2-fixation reaction with propionyl - CoA as a reactant to produce succinyl-CoA. In the absence of adenosyl-B12 or propionyl-CoA, the incorporation of 14CO2 was reduced, although a little radioactivity was observed owing to the endogenous compound. Even in the presence of biotin, the radioactivity did not increase dramatically beyond its level detected in the complete reaction system. Table
Reaction The by
paper
3.
products
formed
reaction
products
chromatography.
detected
in
mixture
incubated
experimental product.
Incorporation
the
into
derived
from
shown
90min. employed.
by cell-free
by
extracts
cell free
of P.
extracts
14C-labeled ƒ¿-ketoglutarate in
corresponding for
succinate
from ƒ¿-ketoglutarate
As
position
conditions
of 14CO2
Fig. to
However, Succinate
7, the
ruber.
of
P.
radioactive
activity was
was also
examined
compound
could
authentic ƒ¿-hydroxyglutaric its
ruber
were
acid not
confirmed
high as
in
under a
reaction
be the the
486
Fig.
S. VEDA,
7. -HG
Paper
chromatogram
,ƒ¿-hydroxyglutaric
of
K. SATO,
reaction
products
acid;ƒ¿-KG,ƒ¿-ketoglutaric
and
S. SHIMIZU
derived acid;
from Succ,
14C-labeled ƒ¿-ketoglutarate.ƒ¿ succinic
acid.
For
detail
of
experimental procedures,seethe text. DISCUSSION In the previous paper (3), we showed that a methanol-utilizing bacterium, P. ruber, could produce a large amount of vitamin B12 in the two different forms, i.e., methyl-B12 and adenosyl-B12. The results concerning the effect of cobalt ion or cyano-B12 on the growth of P. ruber indicate that these two B12 compounds are essential for the growth of this bacterium. When cyano-B12, which was used in this study because of its stability, was added to the cobalt-deficient medium, a slightly longer lag period was observed. This phenomenon might partly be due to some difficulty in the incorporation of cyano-B12 into the cells and partly due to that in the transformation from cyano-B12 into the active forms such as methyl-B12 and adenosyl-B12. In this regard, TAMAOet al. (18) reported that the transformation from cyano-B12 into adenosyl-B12 occurred with some difficulty in the cells of P. shermanii. As mentioned above, P. ruber produces vitamin B12 in the forms of methyl-B12 and adenosyl-B12. This implies that there are at least two different vitamin B12 dependent enzyme systems in the cells of P. ruber. We have already demonstrated that methyl-B12 participates in methionine synthesis from homocysteine and N5 - methyltetrahydrofolate in the cells of P. ruber (3). In spite of the occurrence of B12 - dependent methionine synthetase, methionine had little effect on the growth, so far as examined. Although it is difficult to provide a definite explanation at the present stage of this study, one can speculate that if the absolute amount of cobalt, which is converted into vitamin B12 and required for methionine synthesis in P. ruber, is
V. B12 AND
ITS
RELATED
ENZYME
SYSTEMS
IN P. RUBER
487
small, this bacterium would be able to synthesize methionine with a trace amount of cobalt ion remaining in the inoculum or in the purified medium. This interpretation might be supported by the result that the cells grown on succinate under cobalt deficient conditions had methionine synthetase activity corresponding to about 10% of that in the cells grown under cobalt-sufficient conditions (unpublished data). Further investigation is needed for any final conclusion. On the other hand, adenosyl-B12 is well known to function as the coenzyme of methylmalonyl-CoA mutase in animal tissues and microorganisms such as P. shermanii. As the addition of succinate to the cobalt-deficient medium resulted in the sufficient stimulation of growth, we presumed the existence of methylmalonyl-CoA mutase in the cells of P. ruber. This presumption could be demonstrated from the results that the disappearance of methylmalonyl-CoA used as a reactant was dependent on the presence of adenosyl-B12 and that succinyl-CoA was detected as an enzymatic reaction product in the 14C-labeling experiment. This finding is of interest because of the first observation from methanol-utilizing bacteria. Additionally, this mutase system seems to play an important role in the metabolism of P. ruber because of its fairly specific activity. In this connection BARKER (7) reported that the specific activity of the mutase was in the range of 10 to 100 nmoles/min/mg of protein in P. shermanii, which is known as one of the best producers of this mutase. The mutase activity could be detected in the cells of P. ruber, even when grown on non-C1 compounds as a sole carbon and energy source (Table 2). Furthermore, an almost constant level of its specific activity was found irrespective of the bacterial growth phase. This observation is completely consistent with our previous result as to the variation in the forms of B12 compounds during growth; adenosyl-B12 was found throughout the cultivation period (3). On the basis of these facts, at least part of the adenosyl-B12 formed functions as a coenzyme of methylmalonyl-CoA mutase in P. ruber, and this enzyme system seems to play a fundamental role in the metabolism of this bacterium. However, there may be other adenosyl-B12 - dependent enzyme system(s) apart from methylmalonyl-CoA mutase in P. ruber because of the incomplete effect of succinate on growth.
Fig.
8. in
Proposed Rhodopseudomonas
metabolic
sequence spheroides.
leading
to
the
formation
of succinate
via ƒ¿-hydroxyglutarate
488
S. VEDA,
In was
recent
years,
proposed
the
with
(19).
In
It
was
participated the
to
which
is
results,
the
reverse
we
the
the
of of
course
of
ruber
presence
of
the
the
study
via
this
catalyzed
of
route.
the
propionyl-CoA
this
the
from
8,
present
propionyl
carboxylase
in
P.
ruber.
from ƒ¿-ketoglutarate, dehydrogenase
of
Fig.
glutamate
In
steps
of ƒ¿-hydroxyglutarate
possibility
in
bacterium,
methylmalonyl-CoA
of ƒ¿-hydroxyglutarate
on
illustrated
B12-producing
succinate
P.
formation
reaction
speculated
cycle,
vitamin
adenosyl-B12-dependent
formation
the
observed
acid
and
extracts
implying
we
S. SHIMIZU
tricarboxylic
that
the
cell-free
succinate,
addition,
the
and
during
indicated in
investigation, CoA
of
SATO,
photosynthetic
spheroides,
metabolism mutase
bypass
the
Rhodopseudomonas
K.
bypass
(20).
in
P.
From
these
ruber.
REFERENCES
1)
2)
NISHIO,
N.,
vitamin
B12
YANG,
production.
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