Tài liệu Báo cáo khoa học: The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol pptx

Eur. J. Biochem. 269, 4484–4494 (2002) Ó FEBS 2002

doi:10.1046/j.1432-1033.2002.03151.x

The crystal structure of coenzyme B12-dependent glycerol dehydratase
in complex with cobalamin and propane-1,2-diol
Mamoru Yamanishi1, Michio Yunoki1, Takamasa Tobimatsu1, Hideaki Sato1, Junko Matsui1, Ayako Dokiya1,
Yasuhiro Iuchi1, Kazunori Oe1, Kyoko Suto2, Naoki Shibata2, Yukio Morimoto2, Noritake Yasuoka2
and Tetsuo Toraya1
1

Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Okayama; 2Department of Life Science,
Himeji Institute of Technology, Hyogo, Japan

Recombinant glycerol dehydratase of Klebsiella pneumoniae
was purified to homogeneity. The subunit composition of
the enzyme was most probably a2b2c2. When (R)- and (S)propane-1,2-diols were used independently as substrates, the
rate with the (R)-enantiomer was 2.5 times faster than that
with the (S)-isomer. In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S)isomer was essentially the same or only slightly higher than
that for the (R)-isomer (Km(R)/Km(S) ¼ 1.5). The crystal
structure of glycerol dehydratase in complex with cyanoco˚
balamin and propane-1,2-diol was determined at 2.1 A
resolution. The enzyme exists as a dimer of the abc heterotrimer. Cobalamin is bound at the interface between the a
and b subunits in the so-called Ôbase-onÕ mode with 5,6dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano
group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (b/a)8 barrel that was formed by a

central region of the a subunit. The substrate propane-1,2diol and essential cofactor K+ are bound inside the (b/a)8
barrel above the corrin ring of cobalamin. K+ is heptacoordinated 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 dehydratase. Although racemic propane-1,2-diol was used for crystallization, 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 glycerol.

Adenosylcobalamin is one of the most unique compounds
in nature. It is a water-soluble organometallic compound
possessing a Co–C r bond and serves as a cofactor for
enzymatic radical reactions including carbon skeleton
rearrangements, heteroatom eliminations and intramolecular amino group migrations [1]. Diol dehydratase (EC
4.2.1.28) of Klebsiella oxytoca is an adenosylcobalamin
(AdoCbl1) dependent enzyme that catalyzes the conversions
of 1,2-diols, such as propane-1,2-diol, glycerol, and 1,2ethanediol, to the corresponding aldehydes [2,3] (Fig. 1).
This enzyme has been studied intensively to establish the
mechanism of action of AdoCbl [4–7]. The structure–
function relationship of the coenzyme has also been
investigated extensively with this enzyme [5–8]. Recently,
we have reported the three-dimensional structures of its

complexes with cyanocobalamin [9] and adeninylpentylcobalamin [10] and theoretical calculations of the entire energy
profile along the reaction pathway with a simplified model
[11–13]. In this sense, together with methylmalonyl-CoA
mutase [14], glutamate mutase [15], and class II ribonucleotide reductase [16], diol dehydratase, is one of the most
suitable systems with which to study the structure-based
mechanisms of the AdoCbl-dependent enzymes [17,18].
Glycerol dehydratase (EC 4.2.1.30) catalyzes the same
reaction (Fig. 1) as diol dehydratase [19–21]. Although this
enzyme is isofunctional with diol dehydratase, these two
enzymes bear different physiological roles in the bacterial

metabolisms [6,7]. Selected genera of Enterobacteriaceae,
such as Klebsiella and Citrobacter, produce both glycerol
and diol dehydratases, but the genes for them are independently 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; aD, bD, and cD, a, b, and
c subunits of diol dehydratase; aG, bG, and cG, a, b, and c subunits of
glycerol dehydratase; buffer A, 0.05 M potassium phosphate buffer
(pH 8); IPTG, isopropyl thio-b-D-galactoside.
(Received 11 June 2002, revised 23 July 2002, accepted 25 July 2002)

Keywords: coenzyme B12; adenosylcobalamin; glycerol
dehydratase; crystal structure; radical enzyme catalysis.


Ó FEBS 2002

Structure of B12-dependent glycerol dehydratase (Eur. J. Biochem. 269) 4485

Fig. 1. Conversion of 1,2-diols to the corresponding aldehydes by diol dehydratase.

subunit structures, there are several distinct differences
between them in the following properties: the rate of suicide
inactivation by glycerol, substrate spectrum, monovalent
cation requirement, affinity for cobalamins, and immunochemical cross-reactivity [6,7].
In this paper, we report the method of purifying
recombinant apoglycerol dehydratase from overexpressing
Escherichia coli cells and the crystal structure of glycerol
dehydratase in complex with cyanocobalamin and propane1,2-diol. We intended to explain the above-mentioned
differences between two dehydratases by comparing the
three-dimensional structure of this enzyme with that of diol
dehydratase [9,10].

EXPERIMENTAL PROCEDURES
Materials
Crystalline AdoCbl was a gift from Eizai, Tokyo, Japan.
DEAE-cellulose was purchased from Wako, Osaka, Japan.
The other chemicals were analytical grade reagents.
Preparation of expression plasmids for His6-tagged
glycerol dehydratase and its His6-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¢ and digested with EcoRV 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(aG). 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 NdeI and BglII and ligated with 3.5-kb
ApaI-BglII fragment and 1.5-kb ApaI-NdeI fragment from
pUSI2E(bD) [35] to yield pUSI2E(bG) and pUSI2E(cG),
respectively. Plasmid pUSI2E(bG) was digested with NdeI
and BglII. Resulting 600-bp NdeI-BglII fragment was
inserted into the NdeI-BglII region of pET19b to produce
pET19b(H6bG). A pair of synthetic oligonucleotides,
5¢-TATGGGCAGCAGCCATCATCATCATCATCACA
GCAGCGGCCTGGTGCCGCGCGGCAGCAC-3¢ and
5¢-TAGTGCTGCCGCGCGGCACCAGGCCGCTGCT
GTGATGATGATGATGATGGCTGCTGCCCA-3¢
were hybridized and inserted to the NdeI site of pUSI2E(cG)
to produce pUSI2E(H6Gc). pUSI2E(bG) was digested with
BamHI and BglII. The resulting 0.7-kb DNA fragment was

ligated with BglII-digested pUSI2E(aG) to produce
pUSI2E(aGbG). The 0.5-kb BamHI–BglII fragment from
pUSI2E(H6cG) was ligated with BglII-digested
pUSI2E(H6cG) to produce plasmid pUSI2E(aGbGH6cG).
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 expression plasmid pUSI2E(GD) [28] was aerobically grown at
37 °C in Luria–Burtani (LB) medium containing propane1,2-diol (0.1%) and ampicillin (50 lgỈmL)1) to D600  0.9,
induced with 1 mM isopropyl thio-b-D-galactoside (IPTG)
for 5 h, and harvested by centrifugation. Harvested cells
were resuspended in buffer A (0.05 M potassium phosphate
buffer; pH 8) containing 2 mM phenylmethanesulfonyl
fluoride and disrupted by sonication for 10 min, followed
by centrifugation. Ammonium sulfate was added to the
supernatant to a final concentration of 50% saturation.
After 60 min at 4 °C, the suspension was centrifuged, and
the precipitate was re-dissolved in buffer A. The solution
was subjected to gel filtration on Sepharose 6BÒ, and peak
fractions containing the enzyme were pooled, dialyzed for
12 h against 40 volumes of 1.5 mM potassium phosphate
buffer (pH 8) containing 0.2% propane-1,2-diol with a
buffer exchange, and loaded on to a hydroxyapatite column
which had previously been equilibrated with 2 mM potassium phosphate buffer (pH 8) containing 0.2% propane1,2-diol. After washing the column with 5 mM potassium
phosphate buffer (pH 8) containing 0.2% propane-1,2-diol,
the enzyme was eluted with 13 mM 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 mM
potassium phosphate buffer (pH 8) containing 2% propane-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(aGbGH6cG) was aerobically grown at 30 °C in
terrific broth containing propane-1,2-diol (0.1%) and ampicillin (50 mg mL)1) to D600  0.9, induced with 1 mM
IPTG for 7 h, and harvested by centrifugation. Harvested
cells were resuspended in buffer A containing 2 mM
phenylmethanesulfonyl fluoride and sonicated as described
above. The extract containing His6-tagged enzyme was
mixed with an equal volume of buffer A containing 20 mM
imidazole and 600 mM KCl and loaded on to an


Ó FEBS 2002

4486 M. Yamanishi et al. (Eur. J. Biochem. 269)

Ni-nitrilotriacetate agarose gel (Qiagen GmbH, Germany)
column which had previously been equilibrated with buffer
A containing 10 mM imidazole and 300 mM KCl. After
washing the column with buffer A containing 10 mM
imidazole and 300 mM KCl, the enzyme was eluted with
buffer A containing 50–100 mM imidazole and 300 mM
KCl. After dialysis against 40 volumes of 50 mM TrisHCl
buffer (pH 8) containing 2% propane-1, 2-diol, 150 mM
KCl and 2.5 mM CaCl2, His6-tagged enzyme was digested
with thrombin at 25 °C for 120 min and run through the
Ni-nitrilotriacetate agarose column to remove the His6-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(H6bG), as described
above for glycerol dehydratase.

staining for glycerol dehydratase was performed as described 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
Substrate-free enzyme used for measuring Km values for
substrates was obtained by gel filtration on Sephadex
G-25Ò or dialysis for 3 days against 500 volumes of buffer
A with several changes. One-minute assay was employed for
measurement of Km for glycerol, as glycerol induces suicide
inactivation of the enzyme [39]. Apparent Km values for
substrates and AdoCbl were determined at an AdoCbl
concentration of 15 lM and at a fixed propane-1,2-diol
concentration of 100 mM, respectively.
EPR measurements

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
potent suicide inactivator [39]. One unit of glycerol dehydratase is defined as the amount of enzyme activity that
catalyzes the formation of 1 lmol of propionaldehyde per
minute under the assay conditions. Protein concentration of
crude enzyme was determined by the method of Lowry et al.

[40] with crystalline bovine serum albumin as a standard.
The concentration of purified enzyme was determined by
measuring the absorbance at 280 nm. The molar absorption
coefficient at 280 nm calculated by the method of Gill and
von Hippel [41] for this enzyme is 112 100 M)1 cm)1.

The complex of glycerol dehydratase with adenosylcobinamide 3-imidazolylpropyl phosphate [45] was formed by
incubating apoenzyme (100 units, 4.55 nmol) at 25 °C for
5 min with 50 nmol of the coenzyme analog in 0.65 mL of
buffer A (pH 8) under a nitrogen atmosphere. Propane-1,2diol (50 lmol) was then added. After the mixture had been
incubated at 25 °C for an additional 30 min, the mixture
was quickly frozen in an isopentane bath (cooled to
 )160 °C) and then in a liquid nitrogen bath. The sample
was transferred to an EPR cavity and kept at )130 °C with
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

Separation of the enzyme into components A and B
A purified preparation of the enzyme (80 units) was applied
to a column (bed volume, 2.0 mL) of DEAE cellulose that
had been equilibrated with 10 mM potassium phosphate
buffer (pH 8) containing 10 mM propane-1,2-diol. After
washing the column with 50 mL of 10 mM potassium
phosphate buffer (pH 8), components A and B were eluted
successively with 5 mL of 10 mM potassium phosphate
buffer (pH 8) containing 40 mM KCl and then with 50 mL

of 10 mM potassium phosphate buffer (pH 8) containing
300 mM 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-1,2diol [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

Purified glycerol dehydratase (64 mgỈmL)1) in 20 mM
potassium phosphate buffer (pH 8) containing 2% propane-1,2-diol was converted to the enzymcyanocobalaminỈpropane-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 Quantum4R 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. No
noncrystallographic symmetry (NCS) restraints were
enforced during whole refinements. For adjusting the
positions of atoms, a composite-omit map (2Fo ) Fc) and

an Fo ) Fc map were used.
˚
A data set obtained was up to 2.0 A resolution.
Crystallographic data are listed in Table 1. The a, b and c
subunits of glycerol dehydratase show substantially high


Ó FEBS 2002

Structure of B12-dependent glycerol dehydratase (Eur. J. Biochem. 269) 4487

Table 1. Statistics of data collection and structure determination. The
values in parentheses are for the highest resolution shell.
Data collection

Refinement

X-ray source
Detector
˚
Wavelength (A)
Temperature (K)
Space group
˚
Unit cell (A)
a
b
c
b (°)
˚

Resolution (A)
Measured reflections
Unique reflections
Completeness (%)
Rmerge
˚
Resolution range (A)
Used reflections
Completeness (%)
Rworka
Rfreeb

SPring-8 BL40B2
ADSC Quantum-4R
0.816
100
P21
81.4
108.2
113.1
96.8
2.0
958 394
130 635
99.6 (97.6)
0.097 (0.38)
45.0–2.1
113 453
99.9
0.208

0.248

R-factor=S||Fo| ) |Fc||/S|Fo|. Rwork or the working R-factor is
calculated on the 90% of the observed reflections used for the
refinement. b Rfree or the free R-factor is calculated on the 10% of
reflections excluded from the refinement.
a

homology to the corresponding subunits of diol dehydratase: their identities are 71, 58 and 54%, respectively, and
their similarities 87, 78 and 73%, respectively [28]. We
started the structure determination by the molecular
replacement method with the abc heterotrimer unit of diol
dehydratase from Protein Data Bank (PDB) entry 1DIO [9]
as a reference structure. After a cross-rotation search,
multiple translation searches were performed, and the
monitor and the packing values were checked to determine
the result from the candidates. We concluded that there
is an (abc)2 dimer in an asymmetric unit of the cell. The

calculated VM value was 3.03 A3ặDa)1. (VM ẳ Vcell/ZặMr,
where Vcell 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
(2Fo ) Fc) was calculated. On this map, distinct electron
densities were observed in the positions next to N- and
C-ends of each chain. They could be assigned to certain

amino-acid residues, because the C-terminal three residues
of aD, the N-terminal 45 residues of bD, and the N-terminal
36 residues and the C-terminal three residues of cD were
missing in the reported structure of diol dehydratase [9]. In
addition, aG and bG are longer by one amino acid than aD
in the N-terminal and by three than bD in the C-terminal,
respectively. In the final structure, all residues of the a chain
(Met1–Glu555), all but N-terminal 10 residues of the b
chain (Phe11–Glu194), and all but N-terminal 3 residues of
the c chain (Lys4–Ser141) could be assigned to the electron
density map. In glycerol dehydratase, the numbers of
missing residues were smaller than those of diol dehydratase. We have not determined yet whether these missing
residues are actually truncated by hydrolysis or not visible
because of their high mobility.
After the successive repeats of modeling, energy-minimization 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
Rwork and Rfree 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 RASTER3D [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
by a conventional method. As shown in Table 2, glycerol
dehydratase overexpressed in E. coli was purified by
ammonium sulfate fractionation and chromatography on
Sepharose 6BÒ, hydroxyapatite, and Sephadex G-200Ò

Table 2. Purification of recombinant glycerol dehydratase.
Total activity
(units)

Purification step
Method 1a
Crude extract
(NH4)2SO4 fractionation
Sepharose 6BÒ
Hydroxyapatite
Sephadex G-200Ò
Method 2b
Crude extract
Ni-nitrilotriacetic
Thrombin digested/Ni-nitrilotriacetic
Sepharose 6BÒ
a

Purification from 4.7 g of wet cells.

b

Total protein

(mg)

Specific activity
(mg)1)

Yield
(%)

Purification
(fold)

12 400
11 500
10 500
8760
7780

823
376
185
141
119

15.1
30.6
56.8
62.1
65.4

100

93
85
71
63

1
2.0
3.8
4.1
4.3

46 000
24 100
9770
8450

1920
234
105
70.6

24.0
100
93
120

100
52
21
18

1
4.2
3.9
5.0

Purification from 13 g of wet cells.


4488 M. Yamanishi et al. (Eur. J. Biochem. 269)

(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 Mr 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

Fig. 2. Characterization of purified glycerol dehydratase by PAGE.
SDS/PAGE (A, E) and nondenaturing PAGE (B, F) analyses of the
enzyme at each purification step of method 1 (A, 13.5% gel; B, 7.5%
gel) and method 2 (E, 12% gel; F, 7.5% gel). Resolved components A
and B were also subjected to SDS/PAGE (C) and nondenaturing
PAGE (D). Proteins were stained by Coomassie brilliant blue G.
Molecular mass markers, Sigma SDS-7 L. Positions of the a, b and c
subunits and their complexes are indicated with arrowheads in the
right. H6-a2b2c2 and H6-c represent the His6-tagged a2b2c2 complex
and His6-tagged c subunit, respectively.

Ó FEBS 2002

nondenaturing conditions in the presence of substrate
(Fig. 2B), however, two bands were seen upon protein staining. 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
that the upper band consisted of the a, b and c subunits in a
molar ratio of 1.0 : 1.0 : 1.2. The lower band was composed
of the a and c subunits in a molar ratio of 1.0 : 0.9. When the
purified enzyme was subjected to nondenaturing PAGE on a
Multigel 2/15 gradient gel [44], two bands appeared upon
protein staining, and only the upper one stained upon activity
staining (data not shown). The Mr values for the upper and
lower bands were 220 000 and 200 000, respectively. These
data suggest that the subunit compositions of the proteins in
the upper and lower bands are most likely a2b2c2 (active
apoenzyme, predicted molecular mass of 196 236 Da) and
a2c2 (component B, predicted molecular mass of 153 526
Da), respectively.
In order to confirm this assignment for the lower band,
we attempted to see what happens if component A is added
to the purified preparation of the enzyme. We prepared
components A and B by separation of purified enzyme
upon DEAE–cellulose chromatography in the absence of

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 components 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 contaminated 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-dehydrating 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. Positions 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 (a2c2)
and a trace of contaminating active apoenzyme, a2b2c2. The
bottom major band that had newly appeared has not been
identified yet.
For brevity, we developed a quick purification method
(method 2) for His6-tagged component A and glycerol
dehydratase. His6-tagged enzyme was overexpressed in
E. coli and purified with an Ni-affinity column. After
removal of His6 tag by digestion with thrombin, followed
by passage through the Ni-nitrilotriacetate column, the
enzyme obtained was incubated with excess component A
and run through a Sepharose 6BÒ column. Highest specific
activity of the enzyme (120 mg)1) was obtained by this
simple procedure (Table 2). PAGE analysis under denaturing (Fig. 2E) and nondenaturing conditions (Fig. 2F)
indicated that the purified enzyme was contaminated with

Ó FEBS 2002

Structure of B12-dependent glycerol dehydratase (Eur. J. Biochem. 269) 4489

component B, and that the contaminating component B
recombined with the b subunit (component A) to form
a2b2c2 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
were in reasonable agreement with those reported previously
for the enzyme from K. pneumoniae (Table 3), suggesting
that the recombinant enzyme and the enzyme from
K. pneumoniae are not distinguishable. When (R)- and (S)propane-1,2-diols were used independently as substrates, the
rate with the (R)-enantiomer was 2.5 times faster than that
with the (S)-isomer (Table 3). The affinity of the enzyme for
the (S)-isomer was essentially the same or only slightly
higher than that for the (R)-isomer [Km(R)/Km(S) ¼ 1.5]. This
preference to the (S)-isomer is significantly less marked than
that reported with diol dehydratase [53,54].
EPR spectroscopic evidence for the ‘base-on’ mode
of cobalamin binding
To identify the Co-coordinating base, EPR spectra of the
suicidally inactivated complexes of the enzyme with unlabeled and [imidazole-15N2]-labeled adenosylcobinamide

3-imidazolylpropyl phosphate were compared. The EPR
spectra obtained with these analogs were exactly the same as
those reported for diol dehydratase [46,55] (data not
shown). With the unlabeled imidazolyl analog, each line
of the hyperfine octet (coupling constant, 10.6 mT) showed
superfine splitting into triplets (coupling constant, 1.9 mT).
With the [imidazole-15N2]-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 (A14N) to that with 15N (A15N) was 0.704, which is in
good agreement with the theoretical one that can be
calculated as follows:
A14N =A15N ẳ c14N =c15N ẳ 0:713 theoreticalị
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
dehydratase [46,56], glycerol dehydratase binds AdoCbl in
the so-called Ôbase-onÕ mode. This conclusion is consistent

with the finding of Poppe et al. that p-cresolylcobamide
coenzyme is inactive and serves as an inhibitor for diol and
glycerol dehydratases [57].
Overall structure of glycerol dehydratase in complex
with cobalamin and propane-1,2-diol
The crystal structure of glycerol dehydratase in complex
with cobalamin and propane-1,2-diol was determined at
˚
2.1 A resolution by the molecular replacement method. The

schematic view of the overall structure is shown in Fig. 3A.
The enzyme exists as a dimer of the abc heterotrimer. There
is a noncrystallographic twofold axis around the center of
Fig. 3A. The structure of an abc heterotrimer unit is shown
in Fig. 3B. The central region of the a subunit constitutes
the (b/a)8 barrel, the so-called TIM (triosephosphate
isomerase) barrel. Propane-1,2-diol, a substrate, and K+,
an essential cofactor, are bound inside the barrel. The active
site-cavity is covered by the corrin ring of cobalamin that is
bound on the interface of the a and b subunits. Two a
subunits form dimer a2 to which two b and two c subunits
are bound separately. This structure is quite similar to that
of diol dehydratase [9]. To compare the Ca trace between
glycerol and diol dehydratases, the abc structure of glycerol
dehydratase superimposed on the structure of diol dehydratase 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 Km 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 dehydratase, 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
explains the above-mentioned very low cross-reactivity of
glycerol dehydratase with anti-(diol dehydratase) antiserum
under nondenaturing conditions and its low but distinct
cross-reactivity under denaturing conditions.

Table 3. Kinetic constants for the coenzyme and substrates.
Km (kcat) [mM (s)1)]
Glycerol dehydratase
Recombinant (method 1)
Recombinant (method 2)
Klebsiella pneumoniae enzyme
a

From [39].

b

Km for
AdoCbl (nM)

20a

From [61]. c From [62].

d

Propane-1,2-diol

1.2
1.1 (860)

8
8

Glycerol

0.32
0.32 ± 0.08d (392)

1.50b

0.36c

Mean ± SD, n ¼ 11–14.

(R)-Propane-1,2-diol

(S)-Propane-1,2-diol

0.40 ± 0.09
(604 ± 32)d

0.27 ± 0.10
(244 ± 23)d

4490 M. Yamanishi et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

Fig. 3. Structure of glycerol dehydratase in complex with cobalamin and propane-1,2-diol. (A) Overall structure. One abc heterotrimer unit of the two
was drawn in a schematic model, and the other in a tube model. a, b and c chains are colored in blue, yellow and orange, respectively, darkening
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 Ca
˚
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].

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-dimethylbenzimidazole moiety coordinating to the cobalt atom.
Again, this binding mode is quite similar to that of diol
dehydratase [9]. Crystallographic indication of the base-on
mode of cobalamin binding in class II ribonucleotide
reductase of Lactobacillus leichmannii has also been reported very recently by Drennan and coworkers [16]. The
amino acid residues of diol dehydratase that are hydrogenbonded to the peripheral amide side chains of the corrin ring

[9] are all conserved in glycerol dehydratase as well. In
addition to these conserved residues, the hydroxyl group of
Serb122 is hydrogen-bonded to the amide oxygen of the
g-acetamide side chain of the corrin ring in glycerol
dehydratase (red dotted line in Fig. 4B). In diol dehydratase, the corresponding residue is Prob155 that cannot form
the hydrogen bond. Furthermore, the lengths of the
hydrogen bonds are shorter with five amino acid residues
and longer with four residues in glycerol dehydratase than
those in the diol enzyme. These may be reminiscent of the
fact that the former enzyme binds AdoCbl much more
tightly than the latter enzyme (Table 3).
In the case of glycerol dehydratase, no electron density
of the cyanide ligand was seen even though diffraction data
were collected at 100 K. The Co–N bond distance between
the cobalt atom and N(3) of 5,6-dimethylbenzimidazole in
˚
the glycerol dehydratase-bound cobalamin is 2.48 A. This
value is close to that in the diol dehydratascobalamin


Ó FEBS 2002

Structure of B12-dependent glycerol dehydratase (Eur. J. Biochem. 269) 4491

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. Activesite residues interacting with the substrate
(green) and K+ (cyan) are shown in ball-andstick models. Cobalamin, pink. (B) Residues

hydrogen-bonded to cobalamin. The residues
interacting with cobalamin from distances
˚
˚
shorter by 0.1 A and longer by 0.1 A than
those in diol dehydratase are colored in yellow
and green, respectively. The label ÔBÕ after the
residue number refers to residues of the b
subunit. The red hydrogen bond is present
only glycerol dehydratase. (C) Orientation of
the a-acetamide side chain of pyrrole ring A of
the corrin ring. A possible hydrogen bond
between the a-acetamide side chain and an
amino acid residue is also shown. (a) Glycerol
dehydratascobalamin complex. (b) Diol
dehydratascobalamin complex whose structure was determined at 4 °C (PDB: 1DIO) [9].
(c) Diol dehydratascyanocobalamin complex
(PDB: 1EGM) [10]. (d) Diol dehydratasadeninylpentylcobalamin complex (PDB:
1EEX) [10].

˚
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) and with
˚
adeninylpentylcobalamin (2.22 A) [10]. We assigned the
former as the diol dehydratascob(II)alamin complex,
because no electron density corresponding to the cyano
group was observed [10]. It has been reported that the

Co–CN bond is cleaved by X-ray irradiation during data
collection with diol dehydratase [58]. Kratky and coworkers have reported that free and glutamate mutase-bound
cyanocobalamin is reduced to cob(II)alamin by X-ray
irradiation [59]. Therefore, we believe that the structure
reported in this paper is also that of the glycerol
dehydratascob(II)alamin complex. The dihedral angle of
the northern and southern least-squares planes is 5.5°,
indicating that the corrin ring of the glycerol dehydratasebound cobalamin is also almost planar, as compared with
that of free cyanocobalamin (14.1°). This value is close to
those in the diol dehydratase-bound cobalamins (2.9–5.1°)
[9,10].
Figure 4C indicates the comparison of the position of the
a-acetamide side chain of pyrrole ring A of the corrin ring in
the glycerol dehydratase-bound cobalamin (Fig. 4Ca) with

those in the diol dehydratase-bound cobalamins. It is clear
that the direction of the a-acetamide side chain is very close
to that in the structure of the diol dehydratascobalamin
complex determined at 4 °C [9] (Fig. 4Cb). It seems that this
side chain turns to the opposite direction to the cobalt atom,
depending upon the steric bulk of the upper axial ligand
(CN– or adeninylpentyl group) (Fig. 4Cc,d). Thus, this
offers evidence that the glycerol dehydratase-bound cobalamin exists in a five-coordinated, square-pyramidal complex, suggesting again that the bound cobalamin is actually
cob(II)alamin.
Substrate- and K+-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 NH4+ in the crystallization solution, as

in diol dehydratase [9]. The two hydroxyl groups of
substrate directly coordinate to K+ (Fig. 5A). The O(2)
and O(1) atoms of the substrate are fixed in the active site by
hydrogen bonding with Glua171 and Glna297 and Hisa144


Ó FEBS 2002

4492 M. Yamanishi et al. (Eur. J. Biochem. 269)

Fig. 5. Interaction of glycerol dehydratase with
the substrate. (A) Stereo drawing of a part of
the electron-density map (omit map, 2Fo ) Fc)
contoured for the substrate and nearby amino
acid residues. K+ and propane-1,2-diol are
shown as a cyan CPK model and a green
stick model, respectively. (B) Stereo drawing
of the substrate and nearby residues. Colored
stick model, glycerol dehydratase; gray stick
model, diol dehydratase.

and Aspa336, respectively (Fig. 4A). K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen
atoms of the active-site residues. Such characteristics of the
substrate- and K+-binding sites are quite similar to those
seen in diol dehydratase [9]. Although racemic propane-1,2diol 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 Fo ) Fc 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 electrondensity 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 discrepancy is at present not clear. In contrast, diol dehydratase
prefers the (S)-isomer (Km(R)/Km(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
diol dehydratase [3]. It is well known that diol dehydratase
undergoes the inactivation by glycerol at a faster rate than
glycerol dehydratase [39,60]. It was reported by Bachovchin
et al. that diol dehydratase distinguishes between ÔRÕ and ƠSÕ
binding conformations, the enzym(R)-glycerol complex
being predominantly responsible for the product-forming
reaction, while the enzym(S)-glycerol complex results
primarily in the inactivation reaction [60]. Therefore, the
less marked preference of the glycerol dehydratase toward
the (S)-isomer explains why it is inactivated by glycerol
during catalysis at a slower rate than the diol dehydratase.

ACKNOWLEDGMENTS
We would like to thank Dr Keiko Miura for her kind help in data
collection at the BL40B2 beamline, SPring-8, Japan. We thank Ms.
Yukiko Kurimoto for her assistance in manuscript preparation.

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