Báo cáo khoa học: Molecular basis for specificities of reactivating factors for adenosylcobalamin-dependent diol and glycerol dehydratases pptx

Molecular basis for specificities of reactivating factors
for adenosylcobalamin-dependent diol and glycerol
dehydratases
Hideki Kajiura
1
, Koichi Mori
1
, Naoki Shibata
2
and Tetsuo Toraya
1
1 Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Japan
2 Graduate School of Life Science, University of Hyogo, Japan
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 enzymes that catalyze
adenosylcobalamin (AdoCbl) (coenzyme B
12
)-depen-
dent conversion of 1,2-propanediol, 1,2-ethanediol, and
glycerol to the corresponding aldehydes [1–5]. These
enzymes encoded in the pdu (propanediol
utilization) operon [6–8] and the dha (dihydroxyace-
tone) regulon [9–12], respectively, are involved in pro-
ducing the electron acceptors propionaldehyde and
Keywords
adenosylcobalamin; coenzyme B
12
; diol
dehydratase; glycerol dehydratase;
reactivating factors

Correspondence
T. Toraya, Department of Bioscience and
Biotechnology, Faculty of Engineering,
Okayama University, Tsushima-naka,
Okayama 700–8530, Japan
Fax: +81 86 2518264
Tel: +81 86 2518194
E-mail:
(Received 26 May 2007, revised 17 August
2007, accepted 29 August 2007)
doi:10.1111/j.1742-4658.2007.06074.x
Adenosylcobalamin-dependent diol and glycerol dehydratases are isofunc-
tional enzymes and undergo mechanism-based inactivation by a physiologi-
cal substrate glycerol during catalysis. Inactivated holoenzymes are
reactivated by their own reactivating factors that mediate the ATP-depen-
dent exchange of an enzyme-bound, damaged cofactor for free adenosylco-
balamin through intermediary formation of apoenzyme. The reactivation
takes place in two steps: (a) ADP-dependent cobalamin release and
(b) ATP-dependent dissociation of the resulting apoenzyme–reactivating
factor complexes. The in vitro experiments with purified proteins indicated
that diol dehydratase-reactivating factor (DDR) cross-reactivates the inacti-
vated glycerol dehydratase, whereas glycerol dehydratase-reactivating factor
(GDR) did not cross-reactivate the inactivated diol dehydratase. We inves-
tigated the molecular basis of their specificities in vitro by using purified
preparations of cognate and noncognate enzymes and reactivating factors.
DDR mediated the exchange of glycerol dehydratase-bound cyanocobala-
min for free adeninylpentylcobalamin, whereas GDR cannot mediate
the exchange of diol dehydratase-bound cyanocobalamin for free ade-
ninylpentylcobalamin. As judged by denaturing PAGE, the glycerol dehydra-
tase–DDR complex was cross-formed, although the diol dehydratase–GDR

complex was not formed. There were no specificities of reactivating factors
in the ATP-dependent dissociation of enzyme–reactivating factor complexes.
Thus, it is very likely that the specificities of reactivating factors are
determined by the capability of reactivating factors to form complexes with
apoenzymes. A modeling study based on the crystal structures of enzymes
and reactivating factors also suggested why DDR cross-forms a complex with
glycerol dehydratase, and why GDR does not cross-form a complex with diol
dehydratase.
Abbreviations
AdePeCbl, adeninylpentylcobalamin; AdoCbl, adenosylcobalamin or coenzyme B
12
; CN-Cbl, cyanocobalamin; DDR, diol dehydratase-
reactivating factor; GDR, glycerol dehydratase-reactivating factor; MBTH, 3-methyl-2-benzothiazolinone hydrazone.
5556 FEBS Journal 274 (2007) 5556–5566 ª 2007 The Authors Journal compilation ª 2007 FEBS
b-hydroxypropionaldehyde. They are essential for the
fermentation of 1,2-propanediol and glycerol, respec-
tively [4,13–17] because these carbon sources are more
reduced substrates than the corresponding carbohy-
drates, and oxidation and reduction must be balanced
for the bacterial growth under anaerobic conditions. In
some bacteria, glycerol dehydratase can be substituted
by the isofunctional diol dehydratase, which is induced
at a low level by glycerol [9,14,18].
The mechanism of action of diol dehydratase has
been extensively studied [4,5,19–22]. Diol and glycerol
dehydratases form an adenosyl radical, a catalytic radi-
cal in the active site, by homolysis of the coenzyme
Co-C bond and catalyze the reactions by utilizing the
high reactivity of the radical. The catalytic and inter-
mediate radicals are protected by proteins from un-

desirable side reactions during catalysis (so-called
‘negative catalysis’ [23]). These enzymes tend to
undergo mechanism-based inactivation (suicide inacti-
vation) by certain substrates or coenzyme analogs (for
reviews see [4,5]). Interestingly, both of them are rap-
idly inactivated by a physiological substrate glycerol
during catalysis [2,24,25] or by O
2
in the absence of
substrate [26,27]. The glycerol inactivation of holodiol
dehydratase is a sort of mechanism-based inactivation,
resulting in the disappearance of organic radical inter-
mediate(s) by side reactions and leaving 5¢-deoxyade-
nosine and hitherto unidentified cobalamin at the
active site [24]. The O
2
inactivation in the absence of
substrate results in the formation of hydroxocobalamin
[26] and might be caused by the reaction of adenosyl
radical with oxygen, although the inactivation prod-
ucts derived from it have not yet been identified. These
inactivations are accompanied by the irreversible cleav-
age of the Co-C bond of the coenzyme. The resulting
damaged cofactors remain tightly bound to apoen-
zyme, which brings about the inactivation of enzymes.
The inactivation by glycerol is enigmatic because glyc-
erol is a growth substrate for the bacteria that produce
these enzymes. We found that the glycerol-inactivated
holoenzymes in permeabilized cells of Klebsiella
pneumoniae and Klebsiella oxytoca are rapidly reacti-

vated in situ in the presence of ATP and Mg
2+
(or
Mn
2+
) [28,29]. The inactive complex between enzyme
and cyanocobalamin (CN-Cbl) is also activated in situ
under the same conditions.
We identified two ORFs in the 3¢-flanking region of
the diol dehydratase genes [30] of K. oxytoca as the
genes encoding a reactivating factor for diol dehydra-
tase and named them ddrAB (diol dehydratase-reacti-
vating factor) genes [31]. These genes correspond to
pduGH [32]. We then identified two ORFs in the
proximity of the glycerol dehydratase genes [33] of
K. pneumoniae as the genes encoding a reactivating
factor for glycerol dehydratase and named them gdrAB
(glycerol dehydratase-reactivating factor) genes [34].
Recombinant DdrA and DdrB proteins as well as
GdrA and GdrB form a tight a
2
b
2
complex and actu-
ally function as their reactivating factor – that is, they
reactivate the glycerol-inactivated and O
2
-inactivated
holoenzymes and activate the inactive enzyme–CN-Cbl
complexes in vitro in the presence of AdoCbl, ATP,

and Mg
2+
[35–37]. They (re)activate the complexes by
mediating the ATP-dependent exchange of the enzyme-
bound, adenine-lacking cobalamins for free adenosyl-
cobalamin, an adenine-containing cobalamin through
intermediary formation of apoenzyme. The function of
reactivating factors is to release a tightly bound ade-
nine-lacking cobalamin from the enzymes by a molecu-
lar chaperone-like mechanism of action. It was
established that the reactivation of the inactivated
holoenzyme by the factors takes place in two steps:
(a) ADP-dependent cobalamin release and (b) ATP-
dependent dissociation of the resulting apoenzyme–
reactivating factor complexes. ATP plays a dual role –
as a precursor of ADP for the first step and as an
effector that causes conformational change of factors
into low-affinity forms of enzymes to be reactivated.
The DhaF and DhaG of Citrobacter freundii were also
confirmed to be involved in the reactivation of glycerol
dehydratase [38]. Recently, the crystal structure of
glycerol dehydratase-reactivating factor (GDR) was
reported [39]. We also solved the crystal structure of
diol dehydratase-reactivating factor (DDR) [40]. Based
on their structures and a modeling study, the molecu-
lar mechanism of the release of a damaged cofactor
from inactivated holoenzymes has been proposed [40].
The transient complexes between factors and enzymes,
suggested from biochemical experiments [36,37], have
been postulated to be formed by subunit swapping or

subunit displacement.
Both DDR and GDR have dimeric (ab)
2
structures –
a large subunit (a) with Mr of 64 kDa (DdrA, GdrA,
DhaF) and a small subunit (b) with Mr of 14 kDa
(DdrB) or 12 kDa (GdrB, DhaG) [35–38]. The identi-
ties of amino acid sequences of large and small sub-
units between them are 61% and 30%, respectively
[31,34]. The latter value is considerably lower than
those of the subunits between diol and glycerol dehy-
dratases (more than 50%) [30,33]. The experiments
with permeabilized cells by toluene treatment (so-called
in situ system) indicated that DDR cross-reactivates
the inactivated glycerol dehydratase effectively,
whereas the reverse was not the case – that is, GDR
did not cross-reactivate the inactivated diol
dehydratase [41]. We concluded that GDR is much
H. Kajiura et al. B
12
-dependent dehydratases and reactivating factors
FEBS Journal 274 (2007) 5556–5566 ª 2007 The Authors Journal compilation ª 2007 FEBS 5557
more specific for the dehydratase partner than DDR,
and that a large subunit of the reactivating factors
principally determines the specificity for a dehydratase
[41]; the reason for this, however, remained unclear.
Seifert et al. reported that C. freundii GDR (DhaF–
DhaG complex) cross-reactivates the glycerol-inacti-
vated glycerol dehydratase of K. pneumoniae, but nei-
ther glycerol dehydratase of Clostridium pasteurianum

nor diol dehydratases of K. oxytoca and Salmonella
typhimurium [38].
In this study, we investigated the specificities of reac-
tivating factors for enzymes in vitro using purified
preparations of proteins and attempted to solve why
DDR cross-reactivates the inactivated glycerol dehy-
dratase, and why GDR does not cross-reactivate the
inactivated diol dehydratase. We reached the conclu-
sion that the specificities of reactivating factors are
determined by the capability of reactivating factors to
form complexes with apoenzymes. A modeling study
based on the crystal structures of enzymes and reacti-
vating factors also supported this conclusion. These
results are described here.
Results
Specificities of reactivating factors in the
reactivation of inactivated holoenzymes
The specificities of reactivating factors in the in vitro
reactivation of inactivated holoenzymes were investi-
gated using purified preparations of reactivating factors
and glycerol-inactivated holodiol dehydratase or holo-
glycerol dehydratase. Reactivation of the glycerol-
inactivated holoenzymes was monitored by the recovery
of their 1,2-propanediol-dehydrating activity. As shown
in Fig. 1A, glycerol-inactivated holodiol dehydratase
was rapidly reactivated by DDR in the presence of
AdoCbl, ATP, and Mg
2+
, but was not reactivated by
these either in the presence of GDR or in the absence of

reactivating factors. In contrast, glycerol-inactivated
hologlycerol dehydratase underwent rapid reactivation
by GDR, relatively slow reactivation by DDR, and no
reactivation in the absence of reactivating factors in the
presence of AdoCbl, ATP, and Mg
2+
(Fig. 1B). That
DDR has a broader specificity and that GDR is highly
specific toward a cognate dehydratase are consistent
with previous studies [38,41].
Specificities of reactivating factors in the
activation of inactive enzyme–CN-Cbl complex
The specificities of reactivating factors in the in vitro
activation of inactive enzyme–CN-Cbl complexes were
studied similarly using purified preparations of reacti-
vating factors and enzyme–CN-Cbl complexes. The
enzyme–CN-Cbl complexes can be considered models
of inactivated holoenzymes [35–37] because CN-Cbl is
an adenine-lacking cobalamin that binds tightly to the
active site of the enzymes. Figure 2A shows that the
diol dehydratase–CN-Cbl complex was rapidly acti-
vated by DDR in the presence of AdoCbl, ATP, and
Mg
2+
, but hardly activated either in the presence of
GDR or in the absence of reactivating factors. Con-
versely, the glycerol dehydratase–CN-Cbl complex
underwent rapid activation by GDR, relatively slow
but significant activation by DDR, and essentially no
activation in the absence of reactivating factors under

the same conditions (Fig. 2B). Again, it was demon-
strated that DDR acts on both diol and glycerol dehy-
dratases, whereas GDR is more specific for glycerol
dehydratase. These conclusions are in good agreement
with earlier results [38,41].
Specificities of reactivating factors in the
promotion of exchange of enzyme-bound CN-Cbl
for free adeninylpentylcobalamin (AdePeCbl)
The absolute requirement for free AdoCbl in addition
to ATP and Mg
2+
in both the reactivation of glycerol-
inactivated holoenzymes and the activation of the
enzyme–CN-Cbl complexes strongly indicates that the
Fig. 1. Specificities of reactivating factors in the reactivation of
glycerol-inactivated holodiol dehydratase (A) and hologlycerol de-
hydratase (B). Glycerol-inactivated holoenzymes (0.75 unit) were
incubated at 37 °C for the indicated time without (open squares)
and with 24 lg of DDR (open circles) or GDR (closed circles) in
0.03
M potassium phosphate buffer (pH 8) containing 21 lM AdoCbl
and 1.2
M 1,2-propanediol in the presence of 24 mM ATP ⁄ 24 mM
MgCl
2
, in a total volume of 25 lL. The amount of propionaldehyde
formed was determined as described in the text. The extents of
reactivation of diol dehydratase by DDR and of glycerol by dehydra-
tase by GDR were 82% and 25% for diol and glycerol dehydrata-
ses, respectively.

B
12
-dependent dehydratases and reactivating factors H. Kajiura et al.
5558 FEBS Journal 274 (2007) 5556–5566 ª 2007 The Authors Journal compilation ª 2007 FEBS
reactivation of inactivated holoenzymes and the activa-
tion of the inactive enzyme–CN-Cbl complexes take
place by exchange of the enzyme-bound damaged
cofactor and CN-Cbl, respectively, for free intact co-
enzyme. CN-Cbl and AdePeCbl can be considered
models of the damaged cofactor (adenine-lacking
cobalamin) and intact coenzyme (adenine-containing
cobalamin), respectively. As shown in Fig. 3A, when
the diol dehydratase–CN-Cbl complex was incubated
with free AdePeCbl, ATP, and Mg
2+
in the presence
of DDR, followed by dialysis to remove unbound co-
balamins, the spectrum of the dialyzate indicated that
the enzyme-bound CN-Cbl was displaced by Ade-
PeCbl. Such an exchange did not occur in the presence
of GDR (Fig. 3B) or in the absence of reactivating fac-
tors (Fig. 3A) under the same conditions. In contrast,
upon incubation of the glycerol dehydratase–CN-Cbl
complex with free AdePeCbl, ATP, and Mg
2+
in the
presence of GDR, followed by dialysis, the enzyme-
bound CN-Cbl was displaced by AdePeCbl (Fig. 3F).
Such an exchange occurred in the presence of DDR as
well (Fig. 3E), but not in the absence of reactivating

factors (Fig. 3E) under the same conditions. The
enzyme-bound AdPeCbl did not undergo displacement
by free CN-Cbl under any conditions (Fig. 3C,D,G,H).
It is thus evident that reactivating factor mediates the
exchange of the enzyme-bound, adenine-lacking cobal-
amin for free, adenine-containing cobalamin toward a
cognate dehydratase. In addition, DDR can mediate a
similar exchange with glycerol dehydratase, a non-
cognate enzyme, whereas GDR cannot mediate the
Fig. 3. Specificities of reactivating factors in the promotion of exchange of enzyme-bound CN-Cbl for free AdePeCbl. Diol dehydratase–CN-
Cbl (A,B), glycerol dehydratase–CN-Cbl (E,F), diol dehydratase–AdePeCbl (C,D), and glycerol dehydratase–AdePeCbl (G,H) complexes were
prepared by incubation of apoenzymes (50 units) with 33 l
M CN-Cbl or AdePeCbl at 37 °C for 30 min in 0.2 mL of 0.05 M potassium phos-
phate buffer (pH 8) containing 0.3
M 1,2-propanediol in the dark. The enzyme–CN-Cbl (A,B,E,F) and enzyme–AdePeCbl (C,D,G,H) complexes
were incubated at 37 °C for 30 min without (broken lines in A,C,E,G) and with 1.25 mg of DDR (solid lines in A,C,E,G) or GDR (solid line in
B,D,F,H) in 0.04
M potassium phosphate buffer (pH 8) containing 20 lM AdePeCbl (A,B,E,F) or CN-Cbl (C,D,G,H) and 20 mM ATP ⁄ 20 mM
MgCl
2
, in a total volume of 0.5 mL. The mixtures were then dialyzed at 4 °C for 48 h against 1000 volumes of 0.01 M potassium phosphate
buffer (pH 8) containing 0.3
M 1,2-propanediol with a buffer change. The spectra of dialyzates were measured with a minus cobalamin con-
trol as reference and corrected for dilution.
Fig. 2. Specificities of reactivating factors in the activation of diol
dehydratase–CN-Cbl (A) and glycerol dehydratase–CN-Cbl (B) com-
plexes. The enzyme–CN-Cbl complexes (0.75 unit) were incubated
at 37 °C for the indicated time without (open squares) and with
24 lg of DDR (open circles) or GDR (closed circles) in 0.03
M potas-

sium phosphate buffer (pH 8) containing 21 l
M AdoCbl and 1.2 M
1,2-propanediol in the presence of 24 mM ATP ⁄ 24 mM MgCl
2
,ina
total volume of 25 lL. The amount of propionaldehyde formed was
determined as described in the text. The extents of activation of diol
dehydratase by DDR and of glycerol dehydratase by GDR were 67%
and 65%, respectively.
H. Kajiura et al. B
12
-dependent dehydratases and reactivating factors
FEBS Journal 274 (2007) 5556–5566 ª 2007 The Authors Journal compilation ª 2007 FEBS 5559
exchange of diol dehydratase-bound CN-Cbl for free
AdePeCbl – that is, GDR is more specific for glycerol
dehydratase, the cognate dehydratase.
Specificities of reactivating factors in the
complex formation with dehydratases
DDR and GDR form a complex with apoenzymes of
diol and glycerol dehydratases, respectively, and tran-
sient formation of such complexes seems to result in
the dissociation of a tightly bound damaged cofactor
or adenine-lacking cobalamin [36,37]. To elucidate the
molecular basis of the specificities of reactivating fac-
tors in the reactivation of inactivated holoenzymes and
the activation of inactive enzyme–cobalamin com-
plexes, the possibility of cross-formation of noncognate
enzyme–reactivating factor complexes were examined.
The complex formation was analyzed by nondenatur-
ing PAGE. When apodiol dehydratase was incubated

with reactivating factors either in the presence of ADP
or in the absence of nucleotides, it formed a new major
band with DDR but not with GDR (Fig. 4A,B,
lanes 1 and 2). In the presence of ATP, the new band
did not appear (Fig. 4C, lanes 1 and 2). When apogly-
erol dehydratase was incubated with reactivating fac-
tors, it formed new major bands with either GDR or
DDR in the presence of ADP or in the absence of
nucleotides (Fig. 4A,B, lanes 3 and 4). In the presence
of ATP, the new major band with DDR did not
appear, whereas a part of the new band with GDR
remained (Fig. 4C, lanes 3 and 4). To identify the new
major band formed between glycerol dehydratase and
DDR, the band was analyzed by two-dimensional
PAGE (nondenaturing PAGE in the first dimension
and SDS⁄ PAGE in the second). The analysis provided
all of the a, b, and c subunits of glycerol dehydratase
and the a and b subunits of DDR (Fig. 5C), indicating
that this new band corresponds to a complex between
them. The protein band observed above the band of
DDR a subunit seems to be not an impurity band but
a band due to the insufficient denaturation of glycerol
dehydratase upon SDS ⁄ PAGE in the second dimen-
sion, because the same band was observed when this
enzyme was subjected to SDS ⁄ PAGE without heat
treatment in the sample buffer (data not shown). Such
a band was not observed when diol dehydratase, DDR
or GDR in the sample buffer was applied even without
heat treatment. The bands observed in combinations
ABC

DEF
Fig. 4. Specificities of reactivating factors in the complex formation with dehydratases. Apoenzymes (ApoE) (A–C) or enzyme–CN-Cbl com-
plexes (EÆCN-Cbl) (D–F) (0.35 unit) were incubated at 37 °C for 10 min without and with 15 lg of DDR or GDR in a volume of 6.5 lL, and
the mixtures were further incubated at 37 °C for 10 min in the absence (A,D) and presence of 21 m
M ADP ⁄ 21 mM MgCl
2
(B,E) or 21 mM
ATP ⁄ 21 mM MgCl
2
(C,F) in 35 mM potassium phosphate buffer (pH 8), in a total volume of 7.5 lL. The mixtures were then subjected to non-
denaturing PAGE (5% gel) in the absence (A,D) and presence of 1 m
M ADP ⁄ 1mM MgCl
2
(B,E) or 1 mM ATP ⁄ 1mM MgCl
2
(C,F). Positions
of diol dehydratase (D), glycerol dehydratase (G), DDR (DR), and GDR (GR) are indicated with arrowheads to the right of the gels, and their
complexes 1–4 to the left. Bands 1 and 2 correspond to 1 : 2 and 1 : 1 DD–DDR complexes, respectively, and bands 3 and 4 to GD–GDR
and GD–DDR complexes, respectively. BPB, Bromophenol blue.
B
12
-dependent dehydratases and reactivating factors H. Kajiura et al.
5560 FEBS Journal 274 (2007) 5556–5566 ª 2007 The Authors Journal compilation ª 2007 FEBS
of cognate dehydratases and reactivating factors were
confirmed to be complexes between them (Fig. 5A,B),
in accordance with our previous results [36,37]. When
similar experiments were carried out with the enzyme–
CN-Cbl complexes, essentially the same results as with
apoenzymes were obtained in the presence of ADP
(Fig. 4E), but almost no enzyme–reactivating factor

complexes were formed in the presence of ATP or in
the absence of nucleotides (Fig. 4D,F). These results
are also consistent with published results [36,37]. In
the cases of both DDR and GDR, tightly bound
CN-Cbl is released upon the binding of reactivating
factors to the enzyme–CN-Cbl complexes. Thus, it is
very likely that the specificities of reactivating factors
in the reactivation of inactivated holoenzymes and the
activation of inactive enzymeÆcobalamin complexes are
determined by the capability of reactivating factors to
form complexes with apoenzymes.
Specificities of reactivating factors in the
inhibition of apoenzymes and the reversal by ATP
The specificities of reactivating factors in the complex
formation with dehydratases were further investigated
by inhibition experiments. Apoenzymes of diol and
glycerol dehydratases were strongly inhibited in a time-
dependent manner by pre-incubation with DDR and
GDR, respectively, either in the presence of ADP or in
the absence of nucleotides, in accordance with previous
results [36,37]. ADP alone did not inhibit the enzymatic
activity (data not shown). This inhibition is due to the
complex formation between enzymes and reactivating
factors [36,37]. In contrast, diol dehydratase was not
inhibited at all by pre-incubation with GDR, a noncog-
nate reactivating factor, either in the presence of ADP
or in the absence of nucleotides (Fig. 6B,C), whereas
glycerol dehydratase was strongly inhibited by pre-incu-
bation with DDR either in the presence of ADP or in
the absence of nucleotides (Fig. 6F,G). Again, these

results indicate that diol dehydratase–GDR complex
was not formed, although glycerol dehydratase–DDR
complex was cross-formed. In all cases, the inhibition
by reactivating factors were completely reversed when
assayed in the presence of ATP. This is because the
enzymeÆreactivating factor complexes dissociate into
apoenzymes and reactivating factors in the presence of
ATP [36,37]. No inhibition was observed by the pre-
incubation of apoenzymes and reactivating factors in
the presence of ATP (Fig. 6D,H). Therefore, it can be
concluded that there are no specificities of reactivating
factors in the ATP-dependent dissociation of enzyme–
reactivating factor complexes.
Buried surface areas between the a subunits
of reactivating factors and the b subunits of
dehydratases
To estimate the strengths of interactions between the
a subunits of reactivating factors and the b subunits of
dehydratases, modeling studies were carried out on the
AB C
Fig. 5. Identification of a new band as a noncognate enzymeÆreactivating factor complex by two-dimensional PAGE. Apodiol dehydratase (A)
and apoglycerol dehydratase (B,C) (0.35 unit) were incubated at 37 °C for 10 min with 15 lg of DDR (A,C) or GDR (B) in a volume of 6.5 lL.
The mixtures were further incubated at 37 °C for 10 min in the presence of 21 m
M ADP ⁄ 21 mM MgCl
2
in 35 mM potassium phosphate buf-
fer (pH 8), in a total volume of 7.5 lL. The mixtures were then subjected to nondenaturing PAGE (6% gel) in the presence of 1 m
M
ADP ⁄ 1mM MgCl
2

(first dimension, from left to right), followed by SDS ⁄ PAGE (6 and 14% gels) (second dimension, from top to bottom).
Positions of the subunits of dehydratases and reactivating factors are indicated with arrowheads to the right of the gels. BPB, Bromophenol
blue. Subscripts D, G, DR, and GR are the same as those in the legend to Fig. 4.
H. Kajiura et al. B
12
-dependent dehydratases and reactivating factors
FEBS Journal 274 (2007) 5556–5566 ª 2007 The Authors Journal compilation ª 2007 FEBS 5561
bases of three-dimensional structures of dehydratases
and reactivating factors. When the b subunits of diol
and glycerol dehydratases are superimposed on the
b subunits of DDR and GDR, respectively, the buried
surface areas between the DDR a and diol dehydratase
b subunits and between the GDR a and glycerol dehy-
dratase b subunits are quite similar (710 and 708 A
˚
2
,
respectively). In the model of the glycerol dehydratase–
DDR cross-formed complex, the buried surface area
is 742 A
˚
2
, significantly higher than those of the cog-
nate complexes. On the other hand, in the diol
dehydratase–GDR model, this value is decreased to
698 A
˚
2
.
Discussion

DDR (re)activates both diol and glycerol dehydratases,
but GDR acts only on glycerol dehydratase, a cognate
enzyme, and the a subunit of reactivating factors prin-
cipally determines the specificity for a dehydratase [41].
The molecular basis of these findings has remained
obscure until recently. It was established with DDR
and GDR that the reactivation of inactivated holoen-
zymes and the activation of inactive enzyme–cobala-
min complexes by reactivating factors take place in
two steps: (a) ADP-dependent cobalamin release, and
(b) ATP-dependent dissociation of the resulting apoen-
zyme–reactivating factor complexes [36,37]. ATP serves
as a precursor of ADP in the first step and as an effec-
tor in the second step. ATP and ADP thus function as
a nucleotide switch that modulates the affinity of reac-
tivating factors for the enzymes to be (re)activated. In
this context, one possibility may be that the specifici-
ties of reactivating factors in the reactivation of inacti-
vated holoenzymes and the activation of inactive
enzyme–cobalamin complexes are determined by their
capability to form a complex with apoenzymes of diol
or glycerol dehydratase. The transient formation of
such complexes results in the dissociation of a tightly
bound damaged cofactor or adenine-lacking cobalamin
[36,37]. The modeling study based on the crystal struc-
tures of diol dehydratase and DDR suggested that the
binding of diol dehydratase b subunit to the DDR
a subunit induces steric repulsion between the a sub-
units of enzyme and DDR, leading to the release of a
damaged cofactor from inactivated holoenzymes [40].

Another possibility may be that enzyme–reactivating
factor complexes are formed even with the noncognate
dehydratase, but the specificities of reactivating fac-
tors might be determined by their effectiveness of
Fig. 6. Specificities of reactivating factors in the inhibition of apoenzymes and the reversal by ATP. Apodiol dehydratase (A–D) and apoglycer-
ol dehydratase (E–H) (0.74 unit) were preliminarily incubated without (broken lines in A,E) and with (solid lines) 23 lg of GDR (B–D) and DDR
(F–H), respectively, in the absence (A,B,E,F) and presence of 16 m
M ADP ⁄ 16 mM MgCl
2
(C,G) or 10 mM ATP ⁄ 10 mM MgCl
2
(D,H) in 38 mM
potassium phosphate buffer (pH 8) containing 0.9 M 1,2-propanediol, in a total volume of 15 lL. The mixtures were incubated at 37 °C for
the indicated time periods, and then diluted 600-fold with 50 m
M potassium phosphate buffer (pH 8) containing 2% 1,2-propanediol. AdoCbl
(15 l
M), 1,2-propanediol (0.1 M) and KCl (50 mM) were added to 0.2 mL of the diluted mixtures without (open circles) and with (closed cir-
cles) additional ATP and MgCl
2
(10 mM each) to a total volume of 1 mL. After incubation at 37 °C for 10 min, the amount of propionaldehyde
formed was determined as described in the text.
B
12
-dependent dehydratases and reactivating factors H. Kajiura et al.
5562 FEBS Journal 274 (2007) 5556–5566 ª 2007 The Authors Journal compilation ª 2007 FEBS
nucleotide switch – that is, some complexes are disso-
ciable by the binding of ATP, but others are not. All
the data reported in this paper indicated that the for-
mer possibility is likely, but not the latter. Thus, we
concluded that specificities of reactivating factors are

determined by their capability to form a complex with
apodehydratases. The structure-based subunit swap-
ping or displacement models of DDR and GDR
[39,40] as well as biochemical data with DDR indicates
that the b subunit of DDR is released upon complex
formation between diol dehydratase and DDR. Thus,
the reason why the a subunit of reactivating factors
principally determines the specificity for a dehydratase
also became clear in this study.
Why does DDR cross-form a complex with glycerol
dehydratase, and why does GDR not cross-form a
complex with diol dehydratase? To solve this enigma,
modeling studies were carried out on the bases of
three-dimensional structures of dehydratases and reac-
tivating factors. The following speculations were pos-
sible. As described above, the buried surface areas
between the a subunits of reactivating factors and the
b subunits of dehydratases decrease in the following
order: glycerol dehydratase–DDR > diol dehydra-
tase–DDR ¼ glycerol dehydratase–GDR > diol dehy-
dratase–GDR. This order suggests the order of
relative strengths of interactions between the b sub-
units of enzymes and the a subunits of reactivating
factors and thus seems to reflect on the stability of
complexes. Another factor possibly determining the
specificity is the differences of the surface charge dis-
tribution on the area. However, the surface charge
distributions of DDR and GDR on the area are quite
similar (data not shown). Thus these modeling studies
suggest that the differences of the buried surface areas

between the cognate and cross-formed complexes pro-
vide the most plausible explanation for the above-
mentioned enigma.
Reactivating factors, such as DDR and GDR, may
be not special but rather general for radical B
12
enzymes, because, in general, their holoenzymes tend
to undergo inactivation during catalysis or by oxygen
in the absence of substrate. As suggested from genetic
evidence [42] as well as from fragmentary similarity
with DdrA and GdrA [4], EutA has been identified as
a reactivating factor for ethanolamine ammonia-lyase,
although the details of its mechanism of action have
not yet been reported [43]. Recently, MeaB, a bacte-
rial homolog of MMAA or CblA [44], has been sug-
gested to function in the GTP-dependent assembly of
holomethylmalonyl-CoA mutase and subsequent pro-
tection of radical intermediates during catalysis
[45,46].
Experimental procedures
Materials
Crystalline AdoCbl was a gift from Eisai Co., Ltd. (Tokyo,
Japan). CN-Cbl was obtained from GlaxoSmithKline,
London, UK. AdePeCbl was prepared as described before
[47]. All other chemicals were analytical grade reagents and
used without further purification.
K. oxytoca diol dehydratase and DDR were purified to
homogeneity from overexpressing Escherichia coli JM109
harbouring expression plasmids pUSI2E(DD) [30] and
pUSI2ENd(6 ⁄ 5b) [31], respectively, as reported previously

[35,48]. K. pneumoniae glycerol dehydratase and GDR were
purified to homogeneity from overexpressing E. coli JM109
harbouring expression plasmids pUSI2E(GD) [33] and
E. coli BL21(DE3) harbouring expression plasmids pET
(gdrB-gdrA) [37], respectively, as reported previously [37,49].
Enzyme and protein assays
Activities of diol and glycerol dehydratases were assayed in
the dark by the 3-methyl-2-benzothiazolinone hydrazone
(MBTH) method [50]. The standard reaction mixture con-
taining an appropriate amount of apoenzyme, 15 lm Ado-
Cbl, 0.1 m 1,2-propanediol, 50 mm KCl, and 35 mm
potassium phosphate buffer (pH 8.0), in a total volume of
1.0 mL, was incubated at 37 °C for 10 min. After the reac-
tion was terminated by adding 1 mL of 0.1 m potassium
citrate buffer (pH 3.6), MBTHÆ HCl was added to a final
concentration of 0.9 mm, and the mixture was incubated
again at 37 °C for 15 min. The amount of propionaldehyde
formed was determined by measuring the absorbance at
305 nm. One unit is defined as the amount of enzyme activ-
ity that catalyzes the formation of 1 lmol propionalde-
hydeÆmin
)1
at 37 °C under the standard assay conditions.
Protein concentrations of purified enzymes and reactivat-
ing factors were determined by measuring the absorbance at
280 nm. The molar absorption coefficients at 280 nm, calcu-
lated by the method of Gill & von Hippel [51] from their
deduced amino acid composition and subunit structure,
were 120 500 m
)1

Æcm
)1
for diol dehydratase [35], 112 100
m
)1
Æcm
)1
for glycerol dehydratase [49], 58 140 m
)1
Æcm
)1
for
DDR [35], and 86 500 m
)1
Æcm
)1
for GDR [37], respectively.
Assays of reactivating factors
DDR and GDR activities were assayed by their capability
of reactivating the glycerol-inactivated holoenzymes and
activating the inactive enzyme–CN-Cbl complexes of diol
and glycerol dehydratases, respectively [35–37]. Glycerol-
inactivated holoenzymes were prepared by incubation of
substrate-free apoenzymes (15 units) with 15 lm AdoCbl at
37 °C for 30 min in 50 lL of 0.03 m potassium phosphate
buffer (pH 8) containing 0.3 m glycerol and 0.05 m KCl,
H. Kajiura et al. B
12
-dependent dehydratases and reactivating factors
FEBS Journal 274 (2007) 5556–5566 ª 2007 The Authors Journal compilation ª 2007 FEBS 5563

followed by dialysis at 4 °C for 48 h against 1000 volumes
of 0.05 m potassium phosphate buffer (pH 8) containing
0.3 m 1,2-propanediol with a buffer change. Complexes of
enzymes with CN-Cbl were prepared by incubation of
apoenzymes (15 units) with 11 lm CN-Cbl at 37 °C for
30 min in 0.2 mL of 0.05 m potassium phosphate buffer
(pH 8) containing 0.3 m 1,2-propanediol. In the standard
assays, glycerol-inactivated holoenzymes or inactive
enzyme–CN-Cbl complexes (0.75 unit) was incubated at
37 °C with appropriate amounts of DDR or GDR in
0.03 m potassium phosphate buffer (pH 8) containing
21 lm AdoCbl and 1.2 m 1,2-propanediol in the presence
of 24 mm ATP ⁄ 24 mm MgCl
2
, in a total volume of 25 lL.
The reaction was terminated by adding 25 lL of 0.1 m
potassium citrate buffer (pH 3.6). After removal of precipi-
tate by centrifugation, the reaction mixture was diluted
appropriately to determine the amount of propionaldehyde
using the MBTH method [50].
PAGE
PAGE was performed under nondenaturing conditions as
described by Davis [52] or under denaturing conditions as
described by Laemmli [53]. Protein was stained with Coo-
massie Brilliant Blue G-250. Nondenaturing PAGE of diol
and glycerol dehydratases was performed in the presence of
0.1 m 1,2-propanediol to prevent their subunits from disso-
ciation [54]. In some experiments, ATP or ADP was also
added with MgCl
2

(1 mm each) and KCl (2 mm) to gels
and electrode buffer. Complex formation was analyzed by
nondenaturing PAGE. Enzyme–CN-Cbl complexes were
prepared by incubation of apoenzyme (2.25 units) with
50 lm CN-Cbl at 37 °C for 30 min in 22.5 lLof33mm
potassium phosphate buffer (pH 8).
Modeling studies
The following coordinates were used for the modeling stud-
ies: nucleotide-free DDR [40], 2D0P; nucleotide-free GDR
[39], 1NBW; CN-Cbl-bound diol dehydratase [55], 1EGM;
CN-Cbl-bound glycerol dehydratase [49], 1IWP. Superim-
positions of the diol dehydratase and glycerol dehydratase
b subunits on the DDR and GDR b subunits were per-
formed with the EBI SSM [56] under the pairwise three-
dimensional alignment mode. Buried molecular surface
areas were calculated with the program CNS [57], for which
the probe radius was 1.4 A
˚
, equivalent to the size of a
water molecule. The averaged values between the A–B and
C–D interfaces are used for discussion.
Acknowledgements
This work was supported in part by Grants-in-Aid for
Scientific Research [(B) 13480195 and 17370038 and
Priority Areas 513 to (TT)] from the Japan Society for
Promotion of Science and the Ministry of Education,
Culture, Sports, Science and Technology, Japan, and
the Grant of Natural Sciences Research Assistance
from the Asahi Glass Foundation, Tokyo, Japan.
References

1 Lee HA Jr & Abeles RH (1963) Purification and prop-
erties of dioldehydrase, an enzyme requiring a cobamide
coenzyme. J Biol Chem 238, 2367–2373.
2 Toraya T, Shirakashi T, Kosuga T & Fukui S (1976)
Substrate specificity of coenzyme B
12
-dependent diol
dehydrase: glycerol as both a good substrate and a
potent inactivator. Biochem Biophys Res Commun 69,
475–480.
3 Pawelkiewicz J & Zagalak B (1965) Enzymic conversion
of glycerol into b-hydroxypropionaldehyde in a cell-free
extract from Aerobacter aerogenes. Acta Biochim Pol 12,
207–218.
4 Toraya T (2003) Radical catalysis in coenzyme
B
12
-dependent isomerization (eliminating) reactions.
Chem Rev 103, 2095–2127.
5 Toraya T (2000) Radical catalysis of B
12
enzymes: struc-
ture, mechanism, inactivation, and reactivation of diol
and glycerol dehydratases. Cell Mol Life Sci 57, 106–127.
6 Jeter RM (1990) Cobalamin-dependent 1,2-propanediol
utilization by Salmonella typhimurium. J Gen Microbiol
136, 887–896.
7 Bobik TA, Ailion M & Roth JR (1992) A single regula-
tory gene integrates control of vitamin B
12

synthesis and
propanediol degradation. J Bacteriol 174, 2253–2266.
8 Rondon MR & Escalante-Semerena JC (1992) The poc
locus is required for 1,2-propanediol-dependent tran-
scription of the cobalamin biosynthetic (cob) and pro-
panediol utilization (pdu) genes of Salmonella
typhimurium. J Bacteriol 174, 2267–2272.
9 Forage RG & Foster MA (1982) Glycerol fermentation
in Klebsiella pneumoniae: functions of the coenzyme
B
12
-dependent glycerol and diol dehydratases. J Bacte-
riol 149, 413–419.
10 Forage RG & Lin ECC (1982) dha system mediating
aerobic and anaerobic dissimilation of glycerol in Kle-
bsiella pneumoniae NCIB 418. J Bacteriol 151, 591–599.
11 Ruch FE, Lengeler J & Lin ECC (1974) Regulation of
glycerol catabolism in Klebsiella aerogenes. J Bacteriol
119, 50–56.
12 Seyfried M, Daniel R & Gottschalk G (1996) Cloning,
sequencing, and overexpression of the genes encoding
coenzyme B
12
-dependent glycerol dehydratase of Citro-
bacter freundii. J Bacteriol 178, 5793–5796.
13 Toraya T, Honda S & Fukui S (1979) Fermentation of
1,2-propanediol and 1,2-ethanediol by some genera of
B
12
-dependent dehydratases and reactivating factors H. Kajiura et al.

5564 FEBS Journal 274 (2007) 5556–5566 ª 2007 The Authors Journal compilation ª 2007 FEBS
Enterobacteriaceae, involving coenzyme B
12
-dependent
diol dehydratase. J Bacteriol 139, 39–47.
14 Toraya T, Kuno S & Fukui S (1980) Distribution of
coenzyme B
12
-dependent diol dehydratase and glycerol
dehydratase in selected genera of Enterobacteriaceae and
Propionibacteriaceae. J Bacteriol 141, 1439–1442.
15 Toraya T & Fukui S (1982) Diol dehydratase. In: B
12
(Dolphin, D, ed.), Vol. 2, pp. 233–262. John Wiley &
Sons, New York, NY.
16 Toraya T (1994) Diol dehydratase and glycerol dehydra-
tase, coenzyme B
12
-dependent isozymes. In Metal Ions
in Bioloical Systems (Sigel, H & Sigel, A, eds), Vol. 30,
pp. 217–254. Dekker, New York, NY.
17 Daniel R, Bobik TA & Gottschalk G (1999) Biochemis-
try of coenzyme B
12
-dependent glycerol and diol dehy-
dratases and organization of the encoding genes. FEMS
Microbiol Rev 22, 553–566.
18 Toraya T, Honda S, Kuno S & Fukui S (1978) Coen-
zyme B
12

-dependent diol dehydratase: regulation of apo-
enzyme synthesis in Klebsiella pneumoniae (Aerobacter
aerogenes) ATCC 8724. J Bacteriol 135, 726–729.
19 Abeles RH & Dolphin D (1976) The vitamin B
12
coen-
zyme. Acc Chem Res 9, 114–120.
20 Re
´
tey J, Umani-Ronchi A, Seibl J & Arigoni D (1966)
On the mechanism of the propanediol dehydrase reac-
tion. Experientia 22, 502–503.
21 Re
´
tey J, Umani-Ronchi A & Arigoni D (1966) On the
stereochemistry of the propanediol dehydrase reaction.
Experientia 22, 72–73.
22 Frey PA (1990) Importance of organic radicals in enzy-
matic cleavage of unactivated C–H bonds. Chem Rev
90, 1343–1357.
23 Re
´
tey J (1990) Enzymic reaction selectivity by negative
catalysis or how do enzymes deal with highly reactive
intermediates? Angew Chem Int Ed Engl 29, 355–361.
24 Bachovchin WW, Eagar RG Jr, Moore KW & Richards
JH (1977) Mechanism of action of adenosylcobalamin:
glycerol and other substrate analogues as substrates and
inactivators for propanediol dehydratase – kinetics, ste-
reospecificity, and mechanism. Biochemistry 16 , 1082–

1092.
25 Poznanskaya AA, Yakusheva MI & Yakovlev VA
(1977) Study of the mechanism of action of adenosylco-
balamin-dependent glycerol dehydratase from Aerobac-
ter aerogenes. II. The inactivation kinetics of glycerol
dehydratase complexes with adenosylcobalamin and its
analogs. Biochim Biophys Acta 484, 236–243.
26 Wagner OW, Lee HA Jr, Frey PA & Abeles RH (1966)
Studies on the mechanism of action of cobamide coen-
zymes. Chemical properties of the enzyme-coenzyme
complex. J Biol Chem 241, 1751–1762.
27 Stroinski A, Pawelkiewicz J & Johnson BC (1974) Allo-
steric interactions in glycerol dehydratase. Purification
of enzyme and effects of positive and negative cooper-
ativity for glycerol. Arch Biochem Biophys 162, 321–330.
28 Honda S, Toraya T & Fukui S (1980) In situ reactiva-
tion of glycerol-inactivated coenzyme B
12
-dependent
enzymes, glycerol dehydratase and diol dehydratase.
J Bacteriol 143, 1458–1465.
29 Ushio K, Honda S, Toraya T & Fukui S (1982) The
mechanism of in situ reactivation of glycerol-inacti-
vated coenzyme B
12
-dependent enzymes, glycerol
dehydratase and diol dehydratase. J Nutr Sci
Vitaminol 28, 225–236.
30 Tobimatsu T, Hara T, Sakaguchi M, Kishimoto Y,
Wada Y, Isoda M, Sakai T & Toraya T (1995)

Molecular cloning, sequencing, and expression of the
genes encoding adenosylcobalamin-dependent diol
dehydrase of Klebsiella oxytoca. J Biol Chem 270,
7142–7148.
31 Mori K, Tobimatsu T, Hara T & Toraya T (1997)
Characterization, sequencing, and expression of the
genes encoding a reactivating factor for glycerol-inacti-
vated adenosylcobalamin-dependent diol dehydratase.
J Biol Chem 272, 32034–32041.
32 Bobik TA, Xu Y, Jeter RM, Otto KE & Roth JR
(1997) Propanediol utilization genes (pdu)ofSalmonella
typhimurium: three genes for the propanediol dehydra-
tase. J Bacteriol 179, 6633–6639.
33 Tobimatsu T, Azuma M, Matsubara H, Takatori H,
Niida T, Nishimoto K, Satoh H, Hayashi R & Toraya T
(1996) Cloning, sequencing, and high level expression of
the genes encoding adenosylcobalamin-dependent
glycerol dehydrase of Klebsiella pneumoniae. J Biol Chem
271, 22352–22357.
34 Tobimatsu T, Kajiura H, Yunoki M, Azuma M &
Toraya T (1999) Identification and expression of the
genes encoding a reactivating factor for adenosylcobala-
min-dependent glycerol dehydratase. J Bacteriol 181,
4110–4113.
35 Toraya T & Mori K (1999) A reactivating factor for
coenzyme B
12
-dependent diol dehydratase. J Biol Chem
274, 3372–3377.
36 Mori K & Toraya T (1999) Mechanism of reactivation

of coenzyme B
12
-dependent diol dehydratase by a
molecular chaperone-like reactivating factor. Biochemis-
try 38, 13170–13178.
37 Kajiura H, Mori K, Tobimatsu T & Toraya T (2001)
Characterization and mechanism of action of a reacti-
vating factor for adenosylcobalamin-dependent glycerol
dehydratase. J Biol Chem 276, 36514–36519.
38 Seifert C, Bowien S, Gottschalk G & Daniel R (2001)
Identification and expression of the genes and purifica-
tion and characterization of the gene products involved
in reactivation of coenzyme B
12
-dependent glycerol
dehydratase of Citrobacter freundii. Eur J Biochem 268,
2369–2378.
39 Liao D-I, Reiss L, Turner I Jr & Dotson G (2003)
Structure of glycerol dehydratase reactivase: a new type
of molecular chaperone. Structure 11 , 109–119.
H. Kajiura et al. B
12
-dependent dehydratases and reactivating factors
FEBS Journal 274 (2007) 5556–5566 ª 2007 The Authors Journal compilation ª 2007 FEBS 5565
40 Shibata N, Mori K, Hieda N, Higuchi Y, Yamanishi M
& Toraya T (2005) Release of a damaged cofactor from a
coenzyme B
12
-dependent enzyme: X-ray structures of diol
dehydratase-reactivating factor. Structure 13, 1745–1754.

41 Tobimatsu T, Kajiura H & Toraya T (2000) Specificities
of reactivating factors for adenosylcobalamin-dependent
diol dehydratase and glycerol dehydratase. Arch Micro-
biol 174, 81–88.
42 Kofoid E, Rappleye C, Stojiljkovic I & Roth J (1999)
The 17-gene ethanolamine (eut) operon of Salmonella
typhimurium encodes five homologues of carboxysome
shell proteins. J Bacteriol 181, 5317–5329.
43 Mori K, Bando R, Hieda N & Toraya T (2004) Identifi-
cation of a reactivating factor for adenosylcobalamin-
dependent ethanolamine ammonia-lyase. J Bacteriol
186, 6845–6854.
44 Dobson CM, Wai T, Leclerc D, Wilson A, Wu X,
Dore
´
C, Hudson T, Rosenblatt DS & Gravel RA (2002)
Identification of the gene responsible for the cblA com-
plementation group of vitamin B
12
-responsive methyl-
malonic acidemia based on analysis of prokaryotic gene
arrangements. Proc Natl Acad Sci USA 99, 15554–
15559.
45 Korotkova N & Lidstrom ME (2004) MeaB is a compo-
nent of the methylmalonyl-CoA mutase complex
required for protection of the enzyme from inactivation.
J Biol Chem 279, 13652–13658.
46 Padovani D & Banerjee R (2006) Assembly and protec-
tion of the radical enzyme, methylmalonyl-CoA mutase,
by its chaperone. Biochemistry 45, 9300–9306.

47 Sando GN, Grant ME & Hogenkamp HPC (1976) The
interaction of adeninylalkylcobalamins with ribonucleo-
tide reductase. Biochim Biophys Acta 428, 228–232.
48 Tobimatsu T, Sakai T, Hashida Y, Mizoguchi N, Miyo-
shi S & Toraya T (1997) Heterologous expression, puri-
fication, and properties of diol dehydratase, an
adenosylcobalamin-dependent enzyme of Klebsiella
oxytoca. Arch Biochem Biophys 347, 132–140.
49 Yamanishi M, Yunoki M, Tobimatsu T, Sato H,
Matsui J, Dokiya A, Iuchi Y, Oe K, Suto K, Shibata
N, et al. (2002) The crystal structure of coenzyme B
12
-
dependent glycerol dehydratase in complex with cobala-
min and propane-1,2-diol. Eur J Biochem 269, 4484–
4494.
50 Toraya T, Ushio K, Fukui S & Hogenkamp HPC
(1977) Studies on the mechanism of the adenosylcobala-
min-dependent diol dehydrase reaction by the use of
analogs of the coenzyme. J Biol Chem 252, 963–970.
51 Gill SC & von Hippel PH (1989) Calculation of protein
extinction coefficients from amino acid sequence data.
Anal Biochem 182, 319–326.
52 Davis BJ (1964) Disc electrophoresis. II. Method and
application to human serum proteins. Ann NY Acad Sci
121, 404–427.
53 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
54 Poznanskaya AA, Tanizawa K, Soda K, Toraya T &

Fukui S (1979) Coenzyme B
12
-dependent diol dehydra-
se: purification, subunit heterogeneity, and reversible
association. Arch Biochem Biophys 194, 379–386.
55 Shibata N, Masuda J, Tobimatsu T, Toraya T, Suto K,
Morimoto Y & Yasuoka N (1999) A new mode of B
12
binding and the direct participation of potassium ion in
enzyme catalysis: X-ray structure of diol dehydratase.
Structure 7, 997–1008.
56 Krissinel E & Henrick K (2004) Secondary-structure
matching (SSM), a new tool for fast protein structure
alignment in three dimensions. Acta Cryst D60, 2256–
2268.
57 Bru
¨
nger AT, Adams PD, Clore GM, DeLano WL,
Gros P, Grosse-Kunstleve RW, Jiang J-S, Kuszewski
J, Nilges M, Pannu NS, et al. (1998) Crystallography
& NMR system (CNS): a new software suite for
macromolecular structure determination. Acta Cryst
D54, 905–921.
B
12
-dependent dehydratases and reactivating factors H. Kajiura et al.
5566 FEBS Journal 274 (2007) 5556–5566 ª 2007 The Authors Journal compilation ª 2007 FEBS