The crystal structure of coenzyme B
12
-dependent glycerol dehydratase
in complex with cobalamin and propane-1,2-diol
Mamoru Yamanishi
1
, Michio Yunoki
1
, Takamasa Tobimatsu
1
, Hideaki Sato
1
, Junko Matsui
1
, Ayako Dokiya
1
,
Yasuhiro Iuchi
1
, Kazunori Oe
1
, Kyoko Suto
2
, Naoki Shibata
2
, Yukio Morimoto
2
, Noritake Yasuoka
2
and Tetsuo Toraya
1

dimethylbenzimidazole of the nucleotide moiety coordina-
ting to the cobalt atom. The electron density of the cyano
group was almost unobservable, suggesting that the cyano-
cobalamin was reduced to cob(II)alamin by X-ray irradi-
ation.Theactivesiteisina(b/a)
8
barrel that was formed by a
central region of the a subunit. The substrate propane-1,2-
diol and essential cofactor K
+
are bound inside the (b/a)
8
barrel above the corrin ring of cobalamin. K
+
is hepta-
coordinated by the two hydroxyls of the substrate and five
oxygen atoms from the active-site residues. These structural
features are quite similar to those of diol dehydratase. A
closer contact between the a and b subunits in glycerol
dehydratase may be reminiscent of the higher affinity of the
enzyme for adenosylcobalamin than that of diol dehydra-
tase. Although racemic propane-1,2-diol was used for cry-
stallization, the substrate bound to glycerol dehydratase was
assigned to the (R)-isomer. This is in clear contrast to diol
dehydratase and accounts for the difference between the two
enzymes in the susceptibility of suicide inactivation by gly-
cerol.
Keywords: coenzyme B
12
; adenosylcobalamin; glycerol

metabolisms [6,7]. Selected genera of Enterobacteriaceae,
such as Klebsiella and Citrobacter, produce both glycerol
and diol dehydratases, but the genes for them are inde-
pendently regulated [22–25]: glycerol dehydratase is induced
when Klebsiella pneumoniae grows in the glycerol medium,
whereas diol dehydratase is fully induced when it grows in
the propane-1,2-diol-containing medium, but only slightly
in the glycerol medium. Glycerol dehydratase is a key
enzyme for the dihydroxyacetone (DHA) pathway
[23,26,27], and its genes are located in the DHA regulon
[28,29]. On the other hand, diol dehydratase is a key enzyme
for the anaerobic degradation of 1,2-diols [30,31], and its
genes are located in the pdu operon [32–34]. Furthermore,
although glycerol and diol enzymes are similar in their
Correspondence to T. Toraya, Department of Bioscience and
Biotechnology, Faculty of Engineering, Okayama University,
Tsushima-naka, Okayama 700–8530, Japan.
Fax: + 81 86 2518264, E-mail:
Abbreviations: AdoCbl, adenosylcobalamin; a
D
, b
D
, and c
D
, a, b, and
c subunits of diol dehydratase; a
G
, b
G
, and c

-tagged
glycerol dehydratase and its His
6
-tagged b subunit
DNA segments encoding carboxyl terminal region of the
glycerol dehydratase a subunit was amplified by PCR using
pUSI2E(GD) [28], pfu DNA polymerase (Stratagene) and
pairs of primers 5¢-TCTGAGTGCGGTGGAAGAGATG
ATGAAGCG-3¢ and 5¢-AGATCTTATTCAATGGTGT
CGGGCTGAACC-3¢ anddigestedwithEcoRV and BglII.
Resulting 210-bp fragment was ligated with the 1.5-kb
HindIII-EcoRV fragment from pUSI2E(GD) and pUSI2E
digested with HindIII and BglII to yield pUSI2E(a
G
). DNA
segments encoding the b and c subunits of glycerol
dehydratase were amplified by PCR using pairs of primers
5¢-CATATGCAACAGACAACCCAAATTCAGCCC-3¢
and 5¢-AGATCTTATCACTCCCTTACTAAGTCGATG-3¢
for the b subunit and 5¢-CATATGAGCGAGAAAACCA
TGCGCGTGCAG-3¢ and 5¢-AGATCTTAGCTTCCTTT
ACGCAGCTTATGC-3¢ for the c subunit. The segments
were digested with NdeIandBglII and ligated with 3.5-kb
ApaI-BglII fragment and 1.5-kb ApaI-NdeI fragment from
pUSI2E(b
D
) [35] to yield pUSI2E(b
G
) and pUSI2E(c
G

pUSI2E(H6c
G
) was ligated with BglII-digested
pUSI2E(H6c
G
) to produce plasmid pUSI2E(a
G
b
G
H6c
G
).
Purification of recombinant glycerol dehydratase
Glycerol dehydratase was purified from recombinant
Escherichia coli by a conventional procedure (method 1)
or Ni-nitrilotriacetate affinity chromatography (method 2).
Substrate propane-1,2-diol was added to all the buffers used
throughout the purification steps to minimize dissociation
of the enzyme into components A and B [36]. All operations
were carried out at 0–4 °C.
Method 1. Recombinant E. coli JM109 harboring expres-
sion plasmid pUSI2E(GD) [28] was aerobically grown at
37 °C in Luria–Burtani (LB) medium containing propane-
1,2-diol (0.1%) and ampicillin (50 lgÆmL
)1
)toD
600
 0.9,
induced with 1 m
M

the enzyme was eluted with 13 m
M
potassium phosphate
buffer (pH 8) containing 2% propane-1,2-diol. The eluate
was concentrated and loaded on to a Sephadex G-200Ò
column which had previously been equilibrated with 20 m
M
potassium phosphate buffer (pH 8) containing 2% pro-
pane-1,2-diol. The enzyme was eluted with the same buffer,
and peak fractions containing the enzyme were pooled.
Method 2. Recombinant E. coli JM109 harboring
pUSI2E(a
G
b
G
H
6
c
G
) was aerobically grown at 30 °Cin
terrific broth containing propane-1,2-diol (0.1%) and ampi-
cillin (50 mg mL
)1
)toD
600
 0.9, induced with 1 m
M
IPTG for 7 h, and harvested by centrifugation. Harvested
cells were resuspended in buffer A containing 2 m
M

M
TrisHCl
buffer (pH 8) containing 2% propane-1, 2-diol, 150 m
M
KCl and 2.5 m
M
CaCl
2
, His
6
-tagged enzyme was digested
with thrombin at 25 °C for 120 min and run through the
Ni-nitrilotriacetate agarose column to remove the His
6
-tag
peptide. Because a part of the enzyme had lost the b subunit,
the enzyme solution was concentrated and supplied with
purified b subunit by incubation at 30 °C for 30 min,
followed by Sepharose 6BÒ gel filtration to remove
unbound, excess b subunit. The b subunit was purified from
E. coli BL21 (DE3) carrying pET19b(H6b
G
), as described
above for glycerol dehydratase.
Enzyme and protein assays
Glycerol dehydratase activity was determined by a
3-methyl-2-benzothiazolinone hydrazone method [37] or
an NADH–alcohol dehydrogenase coupled method [38] at
37 °C. Propane-1,2-diol was used as a substrate for routine
assays because glycerol acts as both a good substrate and a

potassium phosphate
buffer (pH 8) containing 40 m
M
KCl and then with 50 mL
of 10 m
M
potassium phosphate buffer (pH 8) containing
300 m
M
KCl, respectively. Five-milliliter fractions were
collected. Neither component alone was active, while the
enzyme activity was restored upon addition of the other
component. Therefore, components A and B were assayed
by adding an excessive amount of one component and
making the other rate-limiting.
PAGE and activity staining of glycerol dehydratase
PAGE was performed under nondenaturing conditions as
described by Davis [42] in the presence of 0.1
M
propane-1,2-
diol [32], or under denaturing conditions as described by
Laemmli [43]. Protein was stained with Coomassie brilliant
blue G-250. Densitometry was carried out by Personal
Scanning Imager PD110 (Molecular Dynamics). Activity
staining for glycerol dehydratase was performed as des-
cribed previously for diol dehydratase [32]. The apparent
molecular weight of the enzyme was estimated by the
nondenaturing PAGE on a Multigel 2–15% gradient gel
(Daiichi Pure Chemicals, Tokyo, Japan) [44].
Kinetic analysis of the enzyme

a cold nitrogen gas flow controlled by a JEOL JES-VT3A
temperature controller. EPR spectra were taken at )130 °C
on JEOL JES-RE3X spectrometer modified with a Gunn
diode X-band microwave unit under the same conditions as
those described for diol dehydratase [46].
Crystallization and data collection
Purified glycerol dehydratase (64 mgÆmL
)1
)in20m
M
potassium phosphate buffer (pH 8) containing 2% pro-
pane-1,2-diol was converted to the enzymeÆcyanocobal-
aminÆpropane-1,2-diol complex by the same method as that
for diol dehydratase [9] except that lauryldimethylamine
oxide was not included. The complex was crystallized by the
sandwich-drop vapor diffusion method at 4 °C. X-ray
diffraction data were collected at 100 K using the Quantum-
4R CCD detector (ADSC) on the BL40B2 beam line at
SPring-8, Japan (Table 1). Reflection data were indexed,
integrated and scaled using the programs Mosflm and
SCALA in the CCP4 suite [47] with DPS [48].
Structure determination and refinement
The structure of the enzyme was determined and refined
using the program
CNS
[49]. The models were built using
Xfit of
XTALVIEW
[50] and checked by
PROCHECK

2
dimer in an asymmetric unit of the cell. The
calculated V
M
value was 3.03 A
˚
3
ÆDa
)1
.(V
M
¼ V
cell
/ZÆM
r
,
where V
cell
and Z are the unit cell volume and the number of
protein molecules per unit cell, respectively).
At this stage, the residues of diol dehydratase were
replaced with the corresponding residues of glycerol
dehydratase. After one set of rigid-body refinement and
simulated annealing were applied, a composite-omit map
(2F
o
) F
c
) was calculated. On this map, distinct electron
densities were observed in the positions next to N- and

zation and simulated annealing, about 900 water molecules
were picked up, and B-factors for all the atoms were refined.
The structure showed good stereochemistry with root-
mean-square (rms) deviations of 0.006 A
˚
from the ideal
bond length and 1.30° from ideal bond angles. The resulting
R
work
and R
free
were 0.208 and 0.248, respectively, in the
resolution range of 45.0–2.1 A
˚
.
Unless otherwise stated, structural figures were created
with
MOLSCRIPT
[51] and
RASTER
3
D
[52].
Accession number
The atomic coordinates have been deposited in the Protein
Data Bank with an accession code of 1IWP.
RESULTS AND DISCUSSION
Purification and characterization of recombinant
glycerol dehydratase
Recombinant nontagged glycerol dehydratase was purified

Sephadex G-200Ò 7780 119 65.4 63 4.3
Method 2
b
Crude extract 46 000 1920 24.0 100 1
Ni-nitrilotriacetic 24 100 234 100 52 4.2
Thrombin digested/Ni-nitrilotriacetic 9770 105 93 21 3.9
Sepharose 6BÒ 8450 70.6 120 18 5.0
a
Purification from 4.7 g of wet cells.
b
Purification from 13 g of wet cells.
Table 1. Statistics of data collection and structure determination. The
values in parentheses are for the highest resolution shell.
Data collection X-ray source SPring-8 BL40B2
Detector ADSC Quantum-4R
Wavelength (A
˚
) 0.816
Temperature (K) 100
Space group P2
1
Unit cell (A
˚
)
a 81.4
b 108.2
c 113.1
b (°) 96.8
Resolution (A
˚

calculated on the 90% of the observed reflections used for the
refinement.
b
R
free
or the free R-factor is calculated on the 10% of
reflections excluded from the refinement.
Ó FEBS 2002 Structure of B
12
-dependent glycerol dehydratase (Eur. J. Biochem. 269) 4487
(method 1). The enzyme was purified 4.3-fold in a yield of
63%. Specific activity was about 65 units/mg. SDS/PAGE
analysis showed that three bands with an M
r
of 61 000 (a),
22 000 (b) and 16 000 (c) (marked with an arrowhead) were
overexpressed in E. coli carrying pUSI2E(GD) (Fig. 2A)
and progressively enriched upon purification, and that only
these subunits were found in the purified preparation of the
enzyme. When the enzyme was electrophoresed under
nondenaturing conditions in the presence of substrate
(Fig. 2B), however, two bands were seen upon protein stain-
ing. The ratio of the upper protein band to the lower one was
estimated to be approximately 2 by densitometric scanning.
Activity staining of the enzyme indicated that the upper band
reconstituted catalytically active holoenzyme with added
AdoCbl, but the lower one did not (data not shown). The
mobility of the upper band was identical with that of active
glycerol dehydratase in the extract of K. pneumoniae ATCC
25955 (data not shown). Two-dimensional PAGE showed

substrate. Recoveries of activity of components A and B
were 24% and 10%, respectively, although weak glycerol
dehydratase activity was observed in the Ôcomponent BÕ
fraction alone. SDS/PAGE analysis showed that compo-
nents A and B contain the b subunit alone and a 1 : 1
mixture of the a and c subunits, respectively (Fig. 2C).
Thus, it was concluded that the inactive protein contam-
inated in the purified enzyme (lower band in Fig. 2B) is
component B. When an excessive amount of component A
was added to the purified enzyme, propane-1,2-diol-dehy-
drating activity increased by 59%. PAGE analysis under
nondenaturing conditions showed that the catalytically
inactive lower band seen in the purified enzyme was
converted to the active upper band upon the addition of
component A (Fig. 2D). Three bands were observed in the
Ôcomponent BÕ fraction upon nondenaturing PAGE. Posi-
tions of the top and middle bands coincided well with the
two bands observed with the purified enzyme. Thus, it was
suggested that the middle and top minor bands of the
Ôcomponent BÕ fraction correspond to component B (a
2
c
2
)
and a trace of contaminating active apoenzyme, a
2
b
2
c
2

2
c
2
and H
6
-c represent the His6-tagged a
2
b
2
c
2
complex
and His6-tagged c subunit, respectively.
4488 M. Yamanishi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
component B, and that the contaminating component B
recombined with the b subunit (component A) to form
a
2
b
2
c
2
that resisted dissociation upon Sepharose 6BÒ
column chromatography. As a result, the enzyme was
purified 5.0-fold in a yield of 18%. This method was
employed for crystallization of glycerol dehydratase.
Kinetic parameters and stereospecificity
of recombinant glycerol dehydratase
Kinetic constants of the purified recombinant glycerol
dehydratase for AdoCbl, propane-1,2-diol, and glycerol

With the [imidazole-
15
N
2
]-labeled analog, on the other
hand, the hyperfine lines (coupling constant, 10.7 mT)
showed superhyperfine splitting into doublets (coupling
constant, 2.7 mT). The ratio of the coupling constant with
14
N(A
14N
)tothatwith
15
N(A
15N
) was 0.704, which is in
good agreement with the theoretical one that can be
calculated as follows:
A
14N
=A
15N
¼ c
14N
=c
15N
¼ 0:713 ðtheoreticalÞ
where c is a gyromagnetic ratio. These lines of evidence
indicated that the axial ligand to Co(II) is the imidazole of
the coenzyme analog. Therefore, it is evident that, like diol

of diol dehydratase [9]. To compare the C
a
trace between
glycerol and diol dehydratases, the abc structure of glycerol
dehydratase superimposed on the structure of diol dehy-
dratase is shown in Fig. 3C with the rms deviation ranges
differently colored. It is clear that deviations of atoms in the
b and c subunits are relatively large, although the rms
deviation of Ca atoms in the a subunit was less than 1.0 A
˚
.
The K
m
values of glycerol dehydratase for AdoCbl is 40–100
times lower than that of diol dehydratase (Table 3). Such
higher affinity of glycerol dehydratase for AdoCbl may be
explained by the closer contact between the a and b subunits
in which cobalamin sits.
Glycerol dehydratase is isofunctional with diol dehydra-
tase, and its amino acid sequences of the a, b and c subunits
are 71, 58 and 54% identical with those of diol dehydratase
[28]. They are immunologically different or only slightly
cross-reactive under nondenaturing conditions [22], but
anti-(K. oxytoca diol dehydratase) antiserum cross-reacted
with K. pneumoniae glycerol dehydratase to some extent
under denaturing conditions (data not shown). As shown in
Fig. 3D, most of the amino acid residues that are not
conserved between these enzymes are located on the surface
of the glycerol dehydratase molecule, whereas the conserved
residues constitute the core part of the enzyme. This fact

d
Klebsiella pneumoniae enzyme 20
a
1.50
b
0.36
c
a
From [39].
b
From [61].
c
From [62].
d
Mean ± SD, n ¼ 11–14.
Ó FEBS 2002 Structure of B
12
-dependent glycerol dehydratase (Eur. J. Biochem. 269) 4489
Cobalamin-binding site and the conformation
of bound cobalamin
Figure 4A depicts the structure of the active site in the (b/a)
8
barrel. Substrate propane-1,2-diol and K
+
are locked in the
active-site cavity that is isolated from a bulk of water by the
corrin ring of cobalamin. Figure 4B shows the structure
around the enzyme-bound cobalamin. The cobalamin
molecule is bound between the a and b subunits in the
so-called Ôbase-onÕ mode ) that is, with the 5,6-dimethyl-

continuously from the N- to C-terminal sides. Eight b strands constituting the (b/a)
8
barrel are drawn in cartoon. Cobalamin, propane-1,2-diol and
K
+
are shown as CPK models colored in pink, green, and cyan, respectively. (B) Structure of the abc heterotrimer unit. (C) Stereoview of the C
a
traces of the abc heterotrimer unit. The corresponding traces of diol dehydratase are drawn in gray. Root mean square deviation (A
˚
) from the diol
dehydratase structure: dark blue, < 0.5; light blue, 0.5–1.0; yellow, 1.0–1.5; orange, 1.5–2.0; red, 2.0–5; pink, > 5. Cobalamin, propane-1,2-diol
and K
+
are shown as ball-and-stick models. (D) Stereo drawing of the distribution of the conserved and different residues. Identical and different
residues are shown in a blue ball-and-stick model and colored CPK models, respectively. Red, different; orange, weakly conserved; yellow, strongly
conserved [63].
4490 M. Yamanishi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
complex (2.50 A
˚
) whose structure was determined at 4 °C
[9] and significantly longer than those in the complexes of
diol dehydratase with cyanocobalamin (2.18 A
˚
)andwith
adeninylpentylcobalamin (2.22 A
˚
) [10]. We assigned the
former as the diol dehydrataseÆcob(II)alamin complex,
because no electron density corresponding to the cyano
group was observed [10]. It has been reported that the

+
-binding sites
Substrate propane-1,2-diol and the essential cofactor K
+
are bound inside the TIM barrel of the a subunit (Fig. 4A).
This suggests that K
+
bound in the active site of glycerol
dehydratase in the presence of substrate is also not
exchangeable with NH
4
+
in the crystallization solution, as
in diol dehydratase [9]. The two hydroxyl groups of
substrate directly coordinate to K
+
(Fig. 5A). The O(2)
andO(1)atomsofthesubstratearefixedintheactivesiteby
hydrogen bonding with Glua171 and Glna297 and Hisa144
Fig. 4. Structures of the active site and the
cobalamin-binding site. (A) Stereo drawing of
the active-site cavity viewed from the direction
parallel to the plane of the corrin ring. Active-
site residues interacting with the substrate
(green) and K
+
(cyan) are shown in ball-and-
stick models. Cobalamin, pink. (B) Residues
hydrogen-bonded to cobalamin. The residues
interacting with cobalamin from distances

+
-binding sites are quite similar to those
seen in diol dehydratase [9]. Although racemic propane-1,2-
diol was used for purification and crystallization, the
(R)-enantiomer is better fitted to the electron-density map
(Fig. 5A). When R-values were compared with (R)- and
(S)-isomers in the active site, the (R)-isomer gave slightly
lower values. Furthermore, when F
o
) F
c
maps were
compared, there was no significant electron density left for
the (R)-isomer, while slight electron density remained for the
(S)-isomer. Thus, we assigned the (R)-isomer to the electron-
density map. The kinetic results, however, indicate that
glycerol dehydratase shows almost equal affinity toward the
(S)- and (R)-isomers (Table 3). The reason for this discrep-
ancy is at present not clear. In contrast, diol dehydratase
prefers the (S)-isomer (K
m(R)
/K
m(S)
¼ 3.1–3.2) [9]. The
subtle differences between glycerol and diol dehydratases
in the positions of Vala301, Sera302, and Aspa336 (Fig. 5B)
might explain the less marked preference of glycerol
dehydratase to the (S)-enantiomer in the substrate binding.
Glycerol serves as a very good substrate as well as a potent
suicide inactivator for both glycerol dehydratase [39] and

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12
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