JOURNAL OF BACTERIOLOGY, Mar. 1980, p.

1439-1442

Vol. 141, No. 3

0021-9193/80/03-1439/04$02.00/0

Distribution of Coenzyme B12-Dependent Diol Dehydratase

and Glycerol Dehydratase in Selected Genera of
Enterobacteriaceae and Propionibacteriaceae
TETSUO TORAYA,* SACHIKO KUNO, AND SABURO FUKUI
Laboratory of Industrial Biochemistry, Department of Industrial Chemistry, Faculty of Engineering, Kyoto

University, Sakyo-Ku, Kyoto 606, Japan

The presence of diol dehydratase and glycerol dehydratase was shown in

several bacteria of Enterobacteriaceae grown anaerobically on 1,2-propanediol
and on glycerol, respectively. Diol dehydratases of Enterobacteriaceae were
immunologically similar, but distinct from that of Propionibacterium freudenreichii.

Vitamin B12 coenzyme-dependent diol dehydratase (1,2-propanediol hydro-lyase, EC

4.2.1.28) and glycerol dehydratase (glycerol hydro-lyase, EC 4.2.1.30) are isofunctional inducible enzymes, both of which catalyze the conversion of 1,2-propanediol, 1,2-ethanediol, and glycerol to propionaldehyde, acetaldehyde, and fihydroxypropionaldehyde, respectively (2, 5, 6,
10, 11, 13, 16). The presence of diol dehydratase
has been demonstrated in vitro only with Klebsiella pneumoniae ATCC 8724 (fornerly Aerobacter aerogenes) (2, 5, 15), ATCC 25955 (formerly Aerobacter aerogenes PZH 572, Warsaw)
(13), and very recently with Propionibacterium
freudenreichii (4). The occurrence of glycerol
dehydratase has been shown in Klebsiellapneumoniae ATCC 25955 (6, 10) and in a strain of
Lactobacillus (11). Recently, we reported that
Klebsiella pneumoniae ATCC 25955 produces
diol dehydratase in a 1,2-propanediol medium
and glycerol dehydratase in a glycerol medium
(13). These two dehydratases are dissimilar in
antigenic properties, in monovalent cation-selectivity pattern and in substrate specificity, suggesting that these dehydratases are isozymes in
that organism (13). The present communication
describes their distribution in some genera of
Enterobacteriaceae and Propionibacteriaceae.
Bacteria were cultivated anaerobically at
30C in a complex medium as described previously (13, 15) and harvested in the late exponential or early stationary phase. The activities of
diol dehydratase and glycerol dehydratase were
assayed by the 3-methyl-2-benzothiazolinone
hydrazone method of Toraya et al. (13, 17). To
determine which enzyme was produced, substrate specificity and immunochemical properties were studied (13). That is, the ratio of glycerol-dehydrating activity to 1,2-propanediol-dehydrating activity (G/P) was measured by the
1-min assay and the 10-min assay (13, 15); the

immunochemical cross-reactivity with rabbit antiserun against homogeneous diol dehydratase

(7) of Klebsiella pneumoniae ATCC 8724 was
estimated by both immunoprecipitation of the
dehydratase activity in crude extracts and Ouchterlony double-diffusion analyses (13, 15).
Since 1,2-propanediol, 1,2-ethanediol, and
glycerol are reduced substrates compared with
carbohydrates, only a limited number of bacteria
could ferment them. Several bacteria of the families Enterobacteriaceae and Propionibacteriaceae were selected for examiiniing their capability to grow on 1,2-propanediol, 1,2-ethanediol, or
glycerol in the presence of yeast extract and
tryptone (complex media) under anaerobic conditions (Table 1), because the fermentation of
glycerol is known to occur readily among bacteria of these families. Among the bacteria
tested, significant growth in a 1,2-propanediol
medium was observed only with Klebsiella
pneumoniae, Citrobacter freundii, Citrobacter
intermedium, and Propionibacterium freudenreichii. The growth on 1,2-ethanediol was found
to be closely related with that on 1,2-propanediol. In a glycerol medium, these bacteria of the
Enterobacteriaceae and all of the propionic acid
bacteria tested grew anaerobically. Although
these diols and triol did not support grpwth of
the other bacteria tested, a small but sfficient
amount of cell material was obtained by largescale cultivation in these complex 1,2-diol and
glycerol media (Table 1) as well as in the basal
complex medium, which permitted determination of the dehydratase activities (see footnote
a to Table 2).
Since both diol dehydratase and glycerol dehydratase are inducible enzymes, 1,2-propanediol-grown and glycerol-grown cells were examined for these dehydratase activities. Cell
extracts of 1,2-propanediol-grown Klebsiella
pneumoniae, Citrobacter freundii, Citrobacter

1439

1440

J. BACTERIOL.

NOTES

TABLE 1. Anaerobic growth of Enterobacteriaceae and Propionibacteriaceae on various substratese

Growth (g of dry cells per liter)'

straiGlucose

and
ftaniam

adGlucose

1,2-Pro-

1,2-Ethanediol
0.05
0.05
0.19
0.18
0.17
0.12
0.07
0.05
0.04
0.02
0.07
0.08
0.04
0.07

Basal
0.05
0.08
0.06
0.05
0.08
0.07
0.43
0.22
0.06
0.39
0.21
0.44
0.18
0.09
0.07
0.41
0.19
0.08
0.06
0.06
0.05
0.16
0.05
0.04
0.06
0.05
0.02
0.02
0.02
0.05
0.07
0.05
0.07
0.18
0.29
Propionibacterium freudenreichii ATCC 6207
0.05
0.43
0.06
Propionibacterium shermaniiLAM 1713
0.05
0.63
0.07
Propionibacterium pentosaceum ATCC 4875
aComposition of the basal complex medium: 5.4 g of KH2PO4, 1.2 g of (NH4)2SO4, 0.4 g of MgSO4 * 7H20, 2.0
g of yeast extract, and 2.0 g of tryptone in 1 liter of tap water (14, 15). The medium was adjusted to pH 7.1 with
KOH. The supplement was 7.6 g of 1,2-propanediol, 9.3 g-of 1,2-ethanediol, 9.2 g of glycerol, or 9.0 g of glucose.
Glucose-grown cells were washed twice with 0.9% KCl and inoculated into fresh medium at an initial
concentration of about 0.002 g of dry cells per liter. Anaerobic cultures were grown in test tubes (30 ml) filled to
capacity and stoppered to exclude air.
b Bacterial strains from ATCC (American Type Culture Coliection), IFO (The Institute of Fermentation,
Osaka, Japan), AKU (The Faculty of Agriculture, Kyoto University, Kyoto, Japan), or IAM (The Institute of
Microbiology, University of Tokyo, Tokyo, Japan).
Applied
c
Maximal growth in 3-day cultivation. Growth was determined by comparing the turbidity of the cultured
broth at 580 nm with standard curves.

Escherichia coli B AKU 0012

Escherichia coli K12 IFO 3301
Citrobacter freundii AKU 0009
Citrobacter intermedium AKU 0010
Klebsiellapneumoniae ATCC 8724
Klebsiellapneumoniae ATCC 25955
Enterobacter cloacae ATCC 13047
Serratia marcescens IFO 3046
Proteus vulgaris IFO 3167
Proteus mirabilis IFO 3849
Erwinia aroideae IFO 12380

intermedium, and Propionibacterium freundenreichii showed the 1,2-propanediol- and glycerol-dehydrating activities with a G/P ratio in
the range of 0.5 to 0.8 in the 1-min assay and 0.1
in the 10-min assay (Table 2). These G/P values
are very similar to those with the purified diol
dehydratase of Klebsiella pneumoniae ATCC
8724 (13). The dehydratase activity in extracts
of 1,2-propanediol-grown Enterobacteriaceae
was completely precipitated by rabbit antiserum
raised against homogeneous diol dehydratase of
Klebsiella pneumoniae ATCC 8724. These kinetic and immunochemical data indicate that
these bacteria produce diol dehydratase when
grown on 1,2-propanediol. As judged from Ouchterlony double-diffusion patterns (some examples are shown in Fig. 1), diol dehydratases of
Enterobacteriaceae are immunologically imilar
or essentially identical to that of Klebsiella
pneumoniae ATCC 8724, since the precipitin
bands of their extracts either formed distinct
spurs or coalesced with that of the purified diol
dehydratase of Klebsiella pneumoniae ATCC
8724. However, only the Propionibacterium
freudenreichii diol dehydratase was not reactive
with antiserum (Table 2) (no precipitin bands
on Ouchterlony double-diffusion analyses [not
shown]), indicating that it is different from enterobacterial diol dehydratases.

0.41
0.40
0.43
0.41
0.51
0.58
0.62
0.39
0.43
0.41
0.39
0.49
1.18
0.64

panediol
0.04

Glycerol

Cell extracts of glycerol-grown Klebsiella

pneumoniae, Citrobacter freundii, and Citrobacter intermedium, except for that of Klebsiella pneumoniae ATCC 8724 strain, showed 1,2propanediol- and glycerol-dehydrating activities
with a G/P ratio of 2.0 to 2.6 in the 1-min assay
and 0.4 to 0.6 in the 10-min assay (Table 2).
These ratios are almost the same as those with
glycerol dehydratase of Klebsiella pneumoniae
ATCC 25955 (13). Furthermore, the dehydratase
activity in these extracts was only partially precipitable by anti-diol dehydratase antiserum.
Thus, it is evident that these bacteria produce
glycerol dehydratase when grown anaerobically
on glycerol. Immunodiffusion patterns provided
additional evidence for the poor cross-reactivity
of glycerol dehydratases with anti-diol dehydratase antiserum: only a faint or essentially no
bands of precipitation were seen between these
extracts and antiserum (Fig. 1). Thus, we conclude that many strains of bacteria of Enterobacteriaceae were able to produce both of the
isozymes, diol dehydratase and glycerol dehydratase, depending upon the growth substrate
(1,2-propanediol or glycerol, respectively). Klebsiella pneumoniae ATCC 8724 was exceptional
and might be a natural mutant which lacks
glycerol dehydratase. This strain produces a single dehydratase (diol dehydratase) in both 1,2-

VOL. 141, 1980

NOTES

propanediol medium and glycerol medium, although the enzyme level in the latter medium is
much lower (15).
The observation that the dehydratase activity
in extracts of glycerol-grown cells was only partially precipitated by anti-diol dehydratase antiserum (Table 2) suggests two possibilities: (i)
a small amount of diol dehydratase is also induced by glycerol or (ii) glycerol dehydratase
itself has an antigenic structure that cross-reacts
partially with anti-diol dehydratase antiserum.
Possibility (i) may be more likely frond the fact
that, in Klebsiella pneumoniae ATCC 8724,
glycerol is also an effective, although poor, inducer for diol dehydratase (15).
The close relationship between growth on 1,2diols or on glycerol and the presence of specific

1441

dehydratases implies that these enzymes play a

role in the fermnentation of these substrates. Recently, we reported that the diol dehydratasecatalyzed reaction is the initial step in the 1,2diol fermentation by Enterobacteriaceae (14).
The metabolic role of glycerol dehydratase may
be to produce a hydrogen acceptor in the glycerol fermentation (1). It has been reported that
fl-hydroxypropionaldehyde serves as one of the
hydrogen acceptors (1, 12). Since glycerol is
more reduced than the corresponding trioses by
two hydrogen atoms, the ability to convert glycerol to a hydrogen acceptor seems to be essential
for the anaerobic metabolism of glycerol (1). The
data in Tables 1 and 2 together supported this
idea and indicated that the organisms which
produce glycerol dehydratase (or diol dehydra-

TABLE 2. Diol dehydratase and glycerol dehydratase in extracts of various organisms after growth with
1,2-propanediol and glycerol
1,2-Propane-

Ognsa
nGrowth
Organis8mand stran
substrateb

diol-dehy-

drating

activityc
(U/mg)

G/P ratiod
1 min

10 min

Enzyme precipitated
with antidiol dehydratase antiserum
(%)'

Citrobacter freundii AKU 0009

100
1.19
0.5
P
42
G
1.48
2.4
1.13
0.7
P
100
Citrobacter intermedium AKU 0010
1.45
2.1
G
20
P
1.06
0.8
0.1
100
Citrobacter intermedium ATCC 8454
G
0.67
2.4
0.4
38
0.7
0.1
100
1.05
P
Klebsiella pneumoniae ATCC 8724
G
0.21
0.7
0.1
100
100
0.7
0.1
P
1.36
Klebsiella pneumoniae ATCC 25955
G
1.30
2.6
0.5
21
1.44
0.7
P
0.1
100
Klebsiella pneumoniae ATCC 8308
G
0.45
2.0
48
0.6
P
2.64
0.6
0.1
100
Klebsiella pneumoniae ATCC 13882
G
0.97
39
2.6
0.6
P
0.30
0.5
0.1
0
Propionibacterium freudenreichii
G
<0.01
_
_d
_
ATCC 6207
a The following bacteria did not show any detectable diol or glycerol dehydratase activity (specific activity
<0.01 U/mg) when cultivated in a complex 1,2-propanediol or glycerol medium: Escherichia coli B AKU 0012
and K12 IFO 3301, Enterobacter cloacae ATCC 13047, Enterobacter aerogenes ATCC 13048 and ATCC 15038,
Serratia marcescens IFO 3046, Proteus vulgaris IFO 3167, Proteus mirabilis IFO 3849, Erwinia aroideae IFO
12380, Propionibacterium shermanii IAM 1713, Propionibacterium pentosaceum ATCC 4875, Clostridium
kluyveri ATCC 8527. (Although some of these bacteria showed only poor growth in complex 1,2-diol or glycerol
media as well as in the basal complex medium as shown in Table 1, an ample amount of cells for determination
of the dehydratase activities was obtained by large-scale cultivation.)
b P, 1,2-Propanediol; G, glycerol.
'Specific activity, micromoles of propionaldehyde formed per minute per milligram of protein at 370C.
d Measured by the 1-min assay or the 10-min assay (13, 15). Since both diol and glycerol dehydratases are
rapidly inactivated by glycerol during catalysis, the 1-min assay should be more useful for obtaining an accurate
G/P ratio of the initial velocities. However, both the 1-min and 10-min assay results are shown here to describe
the kinetic properties of the enzymes. -, Not tested.
' Percentage of the dehydratase activities removed by immunoprecipitation with large excess anti-diol
dehydratase antiserum (50 to 100 pd of antiserum per unit of dehydratase activity). The equivalence point for
the purified diol dehydratase from Klebsiella pneumoniae ATCC 8724 was 12 ud/U. Percent dehydratase
activities remaining after immunoprecipitation shows an average value obtained by both 1-min and 10-min
assays with 1,2-propanediol and glycerol as substrate.

1442

J. BACTERIOL.

NOTES

of glycerol dehydratase when the bacterium is

grown in a complex glycerol medium (similar to
ours). This supports possibility (i) mentioned
above.
LTERATURE CTED

FIG. 1. Ouchterlony double-diffusion patterns of

rabbit antiserum against diol dehydratase of Klebsiella pneumoniae ATCC 8724 with extracts of 1,2propanediol-grown and glycerol-grown cells. Each
well was filed with 44 141 of antiserum or bacterial
extracts containing 0.15 to 0.35 U of activity. (A)
Center well, antiserum; wells 1 and 4, purified diol
dehydratase of Klebsiella pneumoniae ATCC 8724;
well 2, 1,2-propanediol-grown Citrobacter freundii
AKU0009; well 3, 1,2-propanediol-grown Citrobacter
intermedium AKU 0010; well 5, glycerol-grown Citrobacter freundii AKU 0009; well 6, glycerol-grown'
Citrobacter intermedium AKU 0010. (B) Center well,
antiserum; wells 1 and 4, purified diol dehydratase
of Klebsiella pneumoniae ATCC 8724; well 2, 1,2propanediol-grown Klebsiella pneumoniae ATCC
8308; well 3, 1,2-propanediol-grown Klebsiella pneumoniae ATCC 13882; well 5, glycerol-grown Klebsiella pneumoniae ATCC 8308; well 6, glycerol-grown
Klebsiella pneumoniae ATCC 13882.

tase) can ferment glycerol in the absence of

exogenous hydrogen acceptors. Accumulation of

1. Abeles, R. H., A. M. Brownstein, and C. H. Randles.

1960. ,B-Hydroxypropionaldehyde, an intermediate in
the formation of 1,3-propanediol by Aerobacter aerogenes. Biochim. Biophys. Acta 41:530-531.
2. Abeles, R. H., and H. A. Lee, Jr. 1961. An intramolecular4oxidation-reduction requiring a cobamide coenzyme. J. Biol. Chem. 236:2347-2350.
3. Forage, R. G., and M. A. Foster. 1979. Resolution of
the coenzyme Birdependent dehydratases of Kebsiella
sp. and Citrobacter freundu. Biochim. Biophys. Acta

569:249-258.

4. Hosoi, N., K. Morimoto, C. Ozaki, Y. Kitamoto, and

Y. Ichikawa. 1978. Enzyme activities involved in the
metabolism of 1,2-propanediol by Propionibacterium
freudenreichii. J. Ferment. Technol. 56:566-572.
5. Lee, H. A., Jr., and R. H. Abeles. 1963. Purification and
properties of dioldehydrase, an enzyme requiring a cobamide coenzyme. J. Biol. Chem. 238:2367-2373.
6. Pawelkiewicz, J., and B. Zagalak. 1965. Enzymic conversion of glycerol into ,8-hydroxypropionaldehyde in a
cell-free extract from Aerobacter aerogenes. Acta
Biochim. Pol. 12:207-218.
7. Poznanslaja, A. A., K. Tanizawa, K. Soda, T. Toraya,
and S. Fukui. 1979. Coenzyme Bl2-dependent diol dehydrase: purification, subunit heterogeneity, and reversible association. Arch. Biochem. Biophys. 194:379386.
8. Quastel, J. H., and M. Stephenson. 1925. Further observations on the anaerobic growth of bacteria. Biochem. J. 19:660-666.
9. Quastel, J. H., M. Stephenson, and M. D. Whetham.
1925. Some reactions of resting bacteria in relation to
anaerobic growth. Biochem. J. 19:304-317.
10. Schneider, Z., E. G. Larsen, G. Jacobson, B. C. Johnson, and J. Paweldiewicz. 1970. Purification and
properties of glycerol dehydrase. J. Biol. Chem. 245:

1,3-propanediol in the growth medium and reoxidation of NADH to NAD in cell-free extracts
by ,-hydroxypropionaldehyde were demonstrated with Klebsiella pneumoniae, Citrobac3388-3396.
ter freundii, and Citrobacter intermedium (T.
K. L, and M. Sobolov. 1962. A cobamide-reToraya, S. Honda, S. Kuno, and S. Fukui, un- 11. Smiley,
quiring glycerol dehydrase from an acrolein-forming
which
cannot form
published data). Organisms
Lactobacillus. Arch. Biochem. Biophys. 97:538-543.
hydrogen acceptors such as fi-hydroxypropion- 12. Sobolov, M., and K. L. Smiley. 1960. Metabolism of
glycerol by an acrolein-forming lactobacillus. J. Bactealdehyde require the addition of external elecriol. 79:261-266.
tron acceptors, e.g., fumarate or nitrate, to fer- 13. Toraya,
T., and S. Fukui. 1977. Immunochemical eviment glycerol (8, 9). Three species of propionic
dence for the difference between coenzyme Birdependent diol dehydratase and glycerol dehydratase. Eur. J.
acid bacteria tested were able to grow anaero76:285-289.
bically on glycerol in the absence of exogenous 14. Biochem.
Toraya, T., S. Honda, and S. Fukul. 1979. Fermentation
electron acceptors (Table 1), even though none
of 1,2-propanediol and 1,2-ethanediol by some genera of
of them produced glycerol dehydratase or diol
Enterobacteriaeceae, involving coenzyme Bi2dependent diol dehydratase. J. Bacteriol. 139:39-47.
dehydratase in a glycerol medium (Table 2).
and S. Fukui. 1978.
This is reasonable, since oxidation and reduction 15. Toraya, T., S. Honda, S. Kuno,
Coenzyme B12-dependent diol dehydratase: regulation
can be balanced in the propionic acid fermentain
Kkebsiella pneumonnae
of apoenzyme synthesis
tion from glycerol (glycerol and propionic acid
(Aerobacter aerogenes) ATCC 8724. J. Bacteriol. 135:
are in the same oxidation state).
726-729.
After completion of this work, a paper by 16. Toraya, T., T. Shirakashi, T. Kosuga, and S. Fukui.
1976. Substrate specificity of coenzyme B12-dependent
Forage and Foster (3) appeared which reports
diol dehydrase: glycerol as both a good substrate and a
that not only glycerol dehydratase but also diol
potent inactivator. Biochem. Biophys. Res. Commun.
dehydratase is detected electrophoretically in
69:475-480.
and H. P. C. Hogenthe extracts of K. pneumoniae ATCC 25955 17. Toraya, T., K. Ushio, S. Fukui,
kamp. 1977. Studies on the mechanism of the adenogrown in a synthetic glycerol medium. However,
sylcobalamin-dependent diol dehydrase reaction by the
they also showed that the relative amount of
use of analogs of the coenzyme. J. Biol. Chem. 252:963diol dehydratase induced is much less than that
970.