Báo cáo khoa học: Mechanism of the ring contraction process in vitamin B12 biosynthesis by the anaerobe Propionibacterium shermanii under aerobic conditions docx

Mechanism of the ring contraction process in vitamin B
12
biosynthesis by the anaerobe Propionibacterium shermanii
under aerobic conditions
Katsumi Iida, Kuniaki Ohtaka and Masahiro Kajiwara
Department of Medicinal Chemistry, Meiji Pharmaceutical University, Tokyo, Japan
The anaerobic organism Propionibacterium shermanii
was shown to produce vitamin B
12
from d-glucose as a
carbon source under bubbling of N
2
gas in our previ-
ous study [1]. This implies that d-aminolevulinic acid
(ALA, a biosynthetic intermediate of tetrapyrrole) can
be produced from d-glucose via the tricarboxylic acid
cycle, an oxygen-dependent pathway, by P. shermanii.
We were interested to know whether P. shermanii
would be able to utilize oxygen from air instead, if it
were cultured under aerobic conditions.
Different mechanisms have been proposed for the
ring contraction process in the biosynthetic pathway
to vitamin B
12
[2–10]. In P. shermanii fed with
[1-
13
C,1,1,4-
18
O
3

]ALA under anaerobic conditions,
18
O-dilution of the
13
C
18
O-carbonyl oxygen of the
ring A acetamide group of
13
C,
18
O-vitamin B
12
by
oxygen from water in the medium led us to postulate
that the ring contraction, which involves the migration
of ring A, occurs via hydrolysis of our hypothetical
d-lactone, which is proposed from Eschenmoser’s d-lac-
tone [4], formed from the reaction of the ring A acetate
group at C20 with methylation at C1 [5] as shown in
the top row of Fig. 1. On the other hand, the isolation
of factor IV, which was derived from Co-precorrin-4 as
shown in Fig. 2, from P. shermanii cultured under
anaerobic conditions led to the suggestion that ring
contraction involves the migration of ring A after for-
mation of the d-lactone from the ring A acetate group
to C20, following the methylation of Co-precorrin-3 at
C17 [6,7]. In the aerobe Pseudomonas denitrificans,an
Keywords
biosynthesis;

13
C-NMR; d-amino[1-
13
C,1,1,4-
18
O
3
]levulinic acid; Propionibacterium
shermanii; vitamin B
12
Correspondence
K. Iida and M. Kajiwara, Department of
Medicinal Chemistry, Meiji Pharmaceutical
University, 2-522-1 Noshio, Kiyose-shi,
Tokyo 204-8588, Japan
Fax: +81 424 95 8612
Tel: +81 424 95 8611
E-mail: or

(Received 18 April 2007, revised 14 May
2007, accepted 14 May 2007)
doi:10.1111/j.1742-4658.2007.05880.x
The mechanism of the ring contraction process during vitamin B
12
biosyn-
thesis by the anaerobe Propionibacterium shermanii was investigated under
both aerobic and anaerobic conditions by means of feeding experiments
with d-amino[1-
13
C]levulinic acid (a biosynthetic intermediate of tetra-

pyrrole) and d-amino[1-
13
C,1,1,4-
18
O
3
]levulinic acid in combination with
13
C-NMR spectroscopy. We showed that the characteristic mechanism of
the ring contraction process (the generation of precorrin-3x from formation
of the c-lactone from the ring A acetate group at C1 and hydroxylation at
C20 by molecular oxygen catalyzed by CobG, and the migration of ring D
by cleavage of the carbon–oxygen bond at C1 of precorrin-3x) in the aer-
obe Pseudomonas denitrificans was not seen in P. shermanii under aerobic
conditions, and the mechanism of the ring contraction process in P. sher-
manii was the same irrespective of the presence or absence of oxygen.
Abbreviation
ALA, d-aminolevulinic acid.
FEBS Journal 274 (2007) 3475–3481 ª 2007 The Authors Journal compilation ª 2007 FEBS 3475
aerobic feeding experiment with [4-
13
C,1,1,4-
18
O
3
]ALA
showed that precorrin-3x had the c-lactone formed
from the ring A acetate group at C1 and a hydroxyl
group generated from molecular oxygen, catalyzed by
CobG, at C20, whereas an aerobic feeding experiment

with [1-
13
C,1,1,4-
18
O
3
]ALA revealed no
18
O-dilution of
the
13
C
18
O-carbonyl oxygen of the ring A acetate
group of
13
C,
18
O-precorrin-5x methyl ester. These
results led to the suggestion that ring contraction
involved
13
C,
18
O-precorrin-4 formed from the migra-
tion of ring D followed by cleavage of the carbon–oxy-
gen bond of the c -lactone at C1 of
13
C,
18

O-precorrin-
3x [8–10] as shown in the lower row of Fig. 1.
We were interested in the mechanism of cor-
rin formation by the anaerobe P. shermanii in an
oxygen-containing atmosphere. Therefore, we conduc-
ted aerobic feeding experiments with [1-
13
C]ALA
and [1-
13
C,1,1,4-
18
O
3
]ALA in P. shermanii.We
also repeated our previous [1-
13
C]ALA and
[1-
13
C,1,1,4-
18
O
3
]ALA feeding experiments [5] to con-
firm the mechanism of the anaerobic ring contraction
process in P. shermanii for comparison with the result of
the aerobic feeding experiments.
Results and Discussion
Aerobic feeding experiments with [1-

13
C]ALA and
[1-
13
C,1,1,4-
18
O
3
]ALA in P. shermanii
13
C-Vitamin B
12
(5.3 mg) and
13
C,
18
O-vitamin B
12
(4.3 mg) were isolated from cultures of P. shermanii
aerobically cultivated in phosphate buffer containing
[1-
13
C]ALA and [1-
13
C,1,1,4-
18
O
3
]ALA, respectively.
Their purity was judged to be high, based on a compar-

ison of the
1
H-NMR and UV spectra with those of
authentic vitamin B
12
. The magnified
13
C-NMR spec-
tra of
13
C-vitamin B
12
and
13
C,
18
O-vitamin B
12
are
shown in Fig. 3A,B. In Fig. 3A, seven
13
C-enriched
singlet signals (175.347, 175.699, 176.270, 176.395,
177.654, 178.409, and 178.724) can be observed. In
Fig. 3B, seven pairs of
13
C-enriched singlet signals
(175.318 and 175.347, 175.662 and 175.691, 176.233
and 176.263, 176.351 and 176.387, 177.618 and
177.647, 178.358 and 178.394, and 178.687 and

178.716), can be observed.
By comparison of Fig. 3A with Fig. 3B, seven
13
C-enriched singlet signals (175.347, 175.699, 176.270,
Fig. 1. The highlights of previously postulated mechanisms of the ring contraction process in the biosynthesis of vitamin B
12
in the anaerobe
P. shermanii (top row) and the aerobe P. denitrificans (lower row) obtained by previous [1-
13
C,1,1,4-
18
O
3
]ALA feeding experiments.
Ring contraction mechanism of vitamin B
12
K. Iida et al.
3476 FEBS Journal 274 (2007) 3475–3481 ª 2007 The Authors Journal compilation ª 2007 FEBS
176.395, 177.654, 178.409, and 178.724) in Fig. 3A can
be assigned to seven
13
C-amide carbons (C57, C38,
C61, C27, C43, C32, and C50, respectively) derived
from the
13
C-carbonyl carbon of [1-
13
C]ALA. Seven
downfield
13

C-enriched singlet signals (175.347, 175.691,
176.263, 176.387, 177.647, 178.394, and 178.716) of each
pair of signals in Fig. 3B can also be assigned to seven
13
C-amide carbons (C57, C38, C61, C27, C43, C32, and
C50, respectively) derived from
13
CO-carbonyl carbons,
{-
13
C(¼O)-
18
O-} and {-
13
C(¼O)-O-}, present to an
extent of approximately 20% in [1-
13
C,1,1,4-
18
O
3
]ALA.
The other seven
13
C-enriched singlet signals (175.318,
175.662, 176.233, 176.351, 177.618, 178.358, and
178.687), which were shifted upfield from the above
seven
13
C-enriched singlet signals by 2.21–2.76 Hz

owing to the a-isotope effect of
18
O (Table 1), of each
pair of signals in Fig. 3B can be assigned to seven
13
C-
amide carbons (C57, C38, C61, C27, C43, C32, and
C50, respectively,
13
C
18
O-amide carbons) bearing
18
O.
Comparison of the signal intensity or half-width of
the
13
C-enriched signal of the
13
CO-carbonyl carbon
with that of the
13
C
18
O-carbonyl carbon of each
13
C
18
O-amide group of
13

C,
18
O-vitamin B
12
derived
from [1-
13
C,1,1,4-
18
O
3
]ALA in the
13
C-NMR spectrum
(Fig. 3B) gave the
18
O-retention ratio for each
13
C
18
O-
amide group in
13
C,
18
O-vitamin B
12
, as summarized in
Table 1. The average
18

O-retention ratio of the
13
C
18
O-amide groups (C57, C38, C61, C43, C32, and
C50) was 76%. Namely, the
13
C
18
O-carbonyl oxygens
of [1-
13
C,1,1,4-
18
O
3
]ALA used were completely trans-
ferred to the
13
C
18
O-amide oxygens of
13
C,
18
O-vitamin
B
12
, as they have a similar
18

O-ratio. However, the
18
O-retention ratio of the
13
C
18
O-amide group (C27)
of the ring A was 34%, representing a decrease of
58% from the
18
O-retention ratio of the
13
C
18
O-carbo-
nyl oxygen of [1-
13
C,1,1,4-
18
O
3
]ALA.
Our previous anaerobic [1-
13
C]ALA and
[1-
13
C,1,1,4-
18
O

3
]ALA feeding experiments
in P. shermanii [5] repeated
Our previous [1-
13
C]ALA and [1-
13
C,1,1,4-
18
O
3
]ALA
feeding experiments [5] were repeated to investigate the
18
O-retention ratio for each
13
C
18
O-amide group in
13
C,
18
O-vitamin B
12
anaerobically biosynthesized by
P. shermanii.
13
C-Vitamin B
12
(3.3 mg) and

13
C,
18
O-
vitamin B
12
(4.0 mg) were isolated from cultures of
P. shermanii anaerobically cultivated in phosphate
Fig. 2. The mechanisms of the ring contraction process in the biosynthesis of vitamin B
12
postulated on the basis of the isolation of factor IV
from the anaerobe P. shermanii.
K. Iida et al. Ring contraction mechanism of vitamin B
12
FEBS Journal 274 (2007) 3475–3481 ª 2007 The Authors Journal compilation ª 2007 FEBS 3477
buffer containing [1-
13
C]ALA and [1-
13
C,1,1,4-
18
O
3
]ALA, respectively. Their purity was judged to be
high by comparison of the
1
H-NMR and UV spectra
with those of authentic vitamin B
12
. As shown in

Table 1, seven
13
C-enriched singlet signals (175.347,
175.691, 176.270, 176.395, 177.647, 178.401, and
178.724) can be assigned to seven
13
C-amide carbons
(C57, C38, C61, C27, C43, C32, and C50, respectively)
in the
13
C-NMR spectrum of
13
C-vitamin B
12
. Seven
13
C-enriched singlet signals (175.347, 175.699, 176.270,
176.395, 177.655, 178.409, and 178.724), which were
assigned to
13
CO-amide carbons (C57, C38, C61, C27,
C43, C32, and C50, respectively), as well as seven
13
C-enriched singlet signals (175.318, 175.669, 176.241,
176.365, 177.625, 178.380, and 178.694), which were
assigned to
13
C
18
O-amide carbons (C57, C38, C61,

C27, C43, C32, and C50, respectively) shifted upfield
by 2.21 Hz (Table 1) owing to the
18
O a-isotope effect,
can be observed in the
13
C-NMR spectrum of
13
C,
18
O-vitamin B
12
. These
13
C-NMR data for
13
C-
vitamin B
12
and
13
C,
18
O-vitamin B
12
biosynthesized in
anaerobic [1-
13
C]ALA and [1-
13

C,1,1,4-
18
O
3
]ALA feed-
ing experiments were essentially identical to those that
we reported previously [5].
The
18
O-retention ratio for each
13
C
18
O-amide group
in
13
C,
18
O-vitamin B
12
is also summarized in Table 1.
The average
18
O-retention ratio of the
13
C
18
O-amide
groups (C57, C38, C61, C43, C32, and C50) was 81%,
and so the

13
C
18
O-carbonyl oxygens of
[1-
13
C,1,1,4-
18
O
3
]ALA used were completely trans-
ferred to the
13
C
18
O-amide oxygens of
13
C,
18
O-vitamin
B
12
, which have the same
18
O-retention ratio. The
18
O-
retention ratio of the
13
C

18
O-amide group (C27) of the
ring A was 26%, representing a 68% decrement from
Fig. 3. Magnified
13
C-NMR spectra of
13
C-amide carbons of (A)
13
C-vitamin B
12
biosynthesized from [1-
13
C]ALA and (B)
13
C,
18
O-vitamin B
12
aerobically biosynthe-
sized from [1-
13
C,1,1,4-
18
O
3
]ALA by the
anaerobe P. shermanii.
Ring contraction mechanism of vitamin B
12

K. Iida et al.
3478 FEBS Journal 274 (2007) 3475–3481 ª 2007 The Authors Journal compilation ª 2007 FEBS
the
18
O-retention ratio of the
13
C
18
O-carbonyl oxygen
of [1-
13
C,1,1,4-
18
O
3
]ALA.
Mechanism of the ring contraction process in the
biosynthesis of vitamin B
12
by P. shermanii
Two mechanisms of the ring contraction process in
the anaerobe P. shermanii have been suggested [5–7].
One was based on the results of our anaerobic
[1-
13
C,1,1,4-
18
O
3
]ALA feeding experiment in P. sher-

manii [5], shown in the top row of Fig. 1. The other
was based on the isolation of factor IV from anaerobi-
cally cultivated P. shermanii by Scott et al. [6] and
Wang et al. [7], shown in Fig. 2.
18
O-Dilution of the
13
C
18
O-carbonyl (C27) oxygen of the ring A acetamide
group in
13
C,
18
O-vitamin B
12
biosynthesized via these
two postulated anaerobic mechanisms should be
detectable. Meanwhile, another mechanism for the ring
contraction process by the aerobe P. denitrificans had
been proposed from the results of aerobic
[4-
13
C,1,1,4-
18
O
3
]ALA and [1-
13
C,1,1,4-

18
O
3
]ALA feed-
ing experiments. According to this postulated
mechanism in P. denitrificans,no
18
O-dilution of the
13
C
18
O-carbonyl (C27) oxygen of the ring A acetate
group should be observed [8–10], as can be seen in the
lower row of Fig. 1. Therefore, examination of the
13
C
18
O-carbonyl (C27) oxygen of the ring A acetate
group in
13
C,
18
O-vitamin B
12
biosynthesized in
[1-
13
C,1,1,4-
18
O

3
]ALA feeding experiments should
allow us to distinguish between the proposed aerobic
and anaerobic mechanisms of the ring contraction
process.
When the
18
O-retention ratios of the
13
C
18
O-amide
groups of
13
C,
18
O-vitamin B
12
biosynthesized aerobi-
cally and anaerobically in P. shermanii are compared
(Table 1), the
18
O-retention ratios are almost identical.
These results show that vitamin B
12
was biosynthesized
via the same biosynthetic pathways under both aerobic
and anaerobic conditions in this organism. Further-
more, 58% (aerobically) and 68% (anaerobically)
18

O-
retention ratio decrements of the
13
C
18
O-amide (C27)
oxygen in the ring A show that the mechanism of the
ring contraction process in P. shermanii is the same
irrespective of the presence or absence of oxygen.
In other words, although we did not confirm the pres-
ence of CobG in P. shermanii, we can conclude that the
characteristic aerobic process (hydroxylation at C20
by molecular oxygen catalyzed by CobG) does not
occur in P. shermanii, even when molecular oxygen is
present.
Table 1.
13
C-NMR data of
13
C-vitamin B
12
and
13
C,
18
O-vitamin B
12
biosynthesized from [1-
13
C]ALA or [1-

13
C,1,1,4-
18
O
3
]ALA by the anaerobe
P. shermanii under aerobic or anaerobic conditions.
Carbon
c
Origin
d
Conditions of feeding experiments
Aerobic Anaerobic
13
C-B
12
a13
C,
18
O-B
12
b13
C-B
12
a13
C,
18
O-B
12
b

d
C
d
C
Value (Hz)
e
Ratio (%)
f
d
C
d
C
Value (Hz)
e
Ratio (%)
f
50
13
CO 178.724 178.716 – – 178.724 178.724 – –
50
13
C
18
O – 178.687 2.21 78 – 178.694 2.21 82
32
13
CO 178.409 178.394 – – 178.401 178.409 – –
32
13
C

18
O – 178.358 2.76 78 – 178.380 2.21 83
43
13
CO 177.654 177.647 – – 177.647 177.655 – –
43
13
C
18
O – 177.618 2.21 75 – 177.625 2.21 81
27
13
CO 176.395 176.387 – – 176.395 176.395 – –
27
13
C
18
O – 176.351 2.76 34 – 176.365 2.21 26
61
13
CO 176.270 176.263 – – 176.270 176.270 – –
61
13
C
18
O – 176.233 2.21 76 – 176.241 2.21 83
38
13
CO 175.699 175.691 – – 175.691 175.699 – –
38

13
C
18
O – 175.662 2.21 69 – 175.669 2.21 74
57
13
CO 175.347 175.347 – – 175.347 175.347 – –
57
13
C
18
O – 175.318 2.21 77 – 175.318 2.21 82
a13
C-B
12
represents
13
C-vitamin B
12
biosynthesized from [1-
13
C]ALA.
b13
C,
18
O-B
12
represents
13
C,

18
O-vitamin B
12
biosynthesized from
[1-
13
C,1,1,4-
18
O
3
]ALA.
c
Carbon represents carbon position of vitamin B
12
.
d
Origin indicates the origin of
13
C-enriched singlet signals
assigned to
13
C-amide carbons, and
13
CO and
13
C
18
O represent
13
CO-amide and

13
C
18
O-amide groups, respectively.
e
Value indicates upfield
shift owing to
18
O a-isotope effect in hertz.
f
Ratio is the
18
O-retention ratio of
13
C
18
O-amide oxygen in
13
C
18
O-vitamin B
12
(5). These ratios
were calculated from a comparison of the intensities or half-widths of
13
C-enriched signals of
13
CO-amide and
13C18O
-amide carbons in the

13
C-NMR spectrum of
13
C,
18
O-vitamin B
12
.
K. Iida et al. Ring contraction mechanism of vitamin B
12
FEBS Journal 274 (2007) 3475–3481 ª 2007 The Authors Journal compilation ª 2007 FEBS 3479
Conclusions
Aerobic and anaerobic feeding experiments with
[1-
13
C]ALA and [1-
13
C,1,1,4-
18
O
3
]ALA in P. shermanii
were consistent with the anaerobic ring contraction
mechanisms of vitamin B
12
suggested by Kurumaya
et al. [5], Scott et al. [6] and Wang et al. [7], irrespect-
ive of the presence or absence of molecular oxygen.
The characteristic aerobic processes of hydroxylation
at C20 by molecular oxygen catalyzed by CobG, for-

mation of the c-lactone from the ring A acetate group
at C1, and migration of ring D by cleavage of the car-
bon–oxygen bond at C1, observed in P. denitrificans,
do not operate in P. shermanii even in the presence of
oxygen.
Experimental procedures
Organism, chemicals, instruments, and
equipment
The bacterium used was Propionibacterium freudenreichii
ssp. shermanii (ATCC 9614). [1-
13
C]ALA was synthesized
according to our method [11], via the hydrolysis of ethyl
3-ethoxycarbonyl-5-phthalimido[1-
13
C]levulinate produced
from the coupling of ethyl 4-phthalimidoacetoacetate
and ethyl bromo[1-
13
C]acetate derived from sodium
[1-
13
C]acetate (99 atom %
13
C), which was purchased from
Cambridge Isotope Laboratories (Andover, MA, USA).
[1-
13
C,1,1,4-
18

O
3
]ALA was synthesized by heating
[1-
13
C]ALA with [
18
O]water (95–98 atom %
18
O; purchased
from Cambridge Isotope Laboratories) in the presence of
an acidic catalyst in a sealed tube, as we have described
previously [5]. The
18
O-retention ratios at C1 of
[1-
13
C,1,1,4-
18
O
3
]ALA were shown to be {-
13
C(¼
18
O)-
18
O-} ⁄
[{-
13

C(¼
18
O)-O-} and {-
13
C(¼O)-
18
O-}] ⁄ {-
13
C(¼O)-O-} ¼
68 : 26 : 6 by analysis of the
13
C-NMR signals. Authentic
vitamin B
12
was purchased from Glaxo Operations UK Ltd
(Greenford, UK). All other chemicals were of analytical
grade. All
1
H-NMR (300 MHz) and
13
C-NMR (75 MHz)
spectra were recorded on a Varian Gemini-300 spectrometer
(Varian, Inc., Palo Alto, CA, USA). UV spectra were
recorded on a Jasco UVIDEC-610C spectrometer (Jasco
Corp., Tokyo, Japan). The air pump was a Nisso INNO-
b4000 (Nisso Co., Saitama, Japan).
Aerobic or anaerobic feeding experiments with
[1-
13
C]ALA and [1-

13
C,1,1,4-
18
O
3
]ALA in
P. shermanii
Wet cells (approximately 200 g) of P. shermanii were culti-
vated with bubbling of air (aerobic conditions) or with bub-
bling of N
2
gas (anaerobic conditions), and harvested as
previously described [1,5]. These cells, together with a
solution of [1-
13
C]ALA or [1-
13
C,1,1,4-
18
O
3
]ALA (75 mg)
and l-methionine (150 mg) in water (20 mL), which had
been filtered through a membrane filter (0.2 lm), a solution
of 5,6-dimethylbenzimidazole (75 mg) in 80% ethanol
(2 mL), and 50% d-glucose solution (9.6 mL), were added
to 0.07 m sodium phosphate buffer (pH 7.0, 300 mL) con-
taining CoCl
2
.6H

2
O (3 mg) in two conical flasks. These
were incubated at 27 °C with bubbling of air (aerobic con-
ditions) or with bubbling of N
2
gas (anaerobic conditions),
and adjusted to pH 7.0 with 20% Na
2
CO
3
, and 50% d-glu-
cose solution (19.2 mL) was added during the fermentation.
After 3 days,
13
C-vitamin B
12
and
13
C,
18
O-vitamin B
12
were
isolated from the pellet obtained by centrifugation of the
culture broths, using the methods described in our previous
papers [1,5].
13
C-NMR measurements of
13
C-vitamin B

12
and
13
C,
18
O-vitamin B
12
13
C-NMR (75 MHz) spectra were recorded for solutions
of
13
C-vitamin B
12
and
13
C,
18
O-vitamin B
12
in
2
H
2
O with
1,4-dioxane (67.4 p.p.m.) as an internal standard. The spec-
tral width was 18 102.9 Hz with 65 536 data points, which
corresponds to a resolution of 0.28 Hz per point. The deter-
mined 10° pulse width was 2.3 ls, the acquisition time was
1.504 s, the pulse delay time was 0.496 s, and the number
of scans was approximately 15 000. The assignments of

13
C-NMR signals of vitamin B
12
were carried out on the
basis of reported data [1,5].
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