Báo cáo khoa học: Roles of adenine anchoring and ion pairing at the coenzyme B12-binding site in diol dehydratase catalysis pptx

Roles of adenine anchoring and ion pairing at the
coenzyme B
12
-binding site in diol dehydratase catalysis
Ken-ichi Ogura, Shin-ichi Kunita, Koichi Mori, Takamasa Tobimatsu and Tetsuo Toraya
Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Japan
Adenosylcobalamin (AdoCbl) is a cofactor for enzy-
matic radical reactions, including carbon skeleton
rearrangements, heteroatom eliminations, and intramo-
lecular amino group migrations [1–3]. These reactions
involve the migration of a hydrogen atom from one
carbon atom of the substrate to the adjacent carbon
atom [4,5] in exchange for group X, which moves in the
opposite direction [6]. The reactions are initiated by
abstraction of a hydrogen atom from substrates with an
adenosyl radical that is generated in the active site
through homolysis of the cobalt–carbon (Co–C) bond
of AdoCbl [1–3,7,8]. The activation and homolysis of
the Co–C bond upon coenzyme binding to apoenzyme is
therefore considered to be a key step for all the AdoCbl-
dependent reactions. Diol dehydratase (dl-1,2-pro-
panediol hydrolyase; EC 4.2.1.28) is an enzyme that
catalyzes the AdoCbl-dependent conversion of 1,2-diols
and glycerol to the corresponding aldehydes [9,10].
Keywords
adenine anchoring; adenosylcobalamin;
coenzyme B
12
; diol dehydratase; ion pairing
Correspondence
T. Toraya, Department of Bioscience and

Biotechnology, Graduate School of Natural
Science and Technology, Okayama
University, Tsushima-naka, Okayama
700-8530, Japan
Fax: +81 86 251 8264
Tel: +81 86 251 8194
E-mail:
(Received 4 September 2008, revised 8
October 2008, accepted 15 October 2008)
doi:10.1111/j.1742-4658.2008.06745.x
The X-ray structure of the diol dehydratase–adeninylpentylcobalamin com-
plex revealed that the adenine moiety of adenosylcobalamin is anchored in
the adenine-binding pocket of the enzyme by hydrogen bonding of N3
with the side chain OH group of Sera224, and of 6-NH
2
, N1 and N7 with
main chain amide groups of other residues. A salt bridge is formed
between the e-NH
2
group of Lysb135 and the phosphate group of cobala-
min. To assess the importance of adenine anchoring and ion pairing,
Sera224 and Lysb135 mutants of diol dehydratase were prepared, and their
catalytic properties investigated. The Sa224A, Sa224N and Kb135E
mutants were 19–2% as active as the wild-type enzyme, whereas the
Kb135A, Kb135Q and Kb135R mutants retained 58–76% of the wild-type
activity. The presence of a positive charge at the b135 residue increased
the affinity for cobalamins but was not essential for catalysis, and the
introduction of a negative charge there prevented the enzyme–cobalamin
interaction. The Sa224A and Sa224N mutants showed a k
cat

⁄ k
inact
value
that was less than 2% that of the wild-type, whereas for Lysb135 mutants
this value was in the range 25–75%, except for the Kb135E mutant (7%).
Unlike the wild-type holoenzyme, the Sa224N and Sa224A holoenzymes
showed very low susceptibility to oxygen in the absence of substrate. These
findings suggest that Sera224 is important for cobalt–carbon bond activa-
tion and for preventing the enzyme from being inactivated. Upon inactiva-
tion of the Sa224A holoenzyme during catalysis, cob(II)alamin
accumulated, and a trace of doublet signal due to an organic radical disap-
peared in EPR. 5¢-Deoxyadenosine was formed from the adenosyl group,
and the apoenzyme itself was not damaged. This inactivation was thus
considered to be a mechanism-based one.
Abbreviations
AdePeCbl, adeninylpentylcobalamin; AdoCbl, adenosylcobalamin or coenzyme B
12
; aqCbl, aquacobalamin; CN-Cbl, cyanocobalamin; OH-Cbl,
hydroxocobalamin.
6204 FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS
Structure–function studies using adenine-modified
analogs of AdoCbl have shown relatively low specific-
ity of diol dehydratase for the adenine moiety in the
adenosyl group, an upper axial ligand [11–13]. 1-Deaza
and 3-deaza analogs of AdoCbl are partially active
(56% and 46%, respectively) as coenzyme, whereas
7-deaza and N
6
,N
6

-dimethyl derivatives do not show
detectable coenzyme activity and act as strong compet-
itive inhibitors of AdoCbl. Guanosylcobalamin is an
inactive coenzyme with low affinity for the enzyme.
The nucleotide loop moiety of AdoCbl is not directly
involved in the catalytic process, but it is obligatory
for the continuous progress of catalytic cycles [14–16].
Adenosylcobinamide methyl phosphate, an analog of
AdoCbl lacking the nucleotide loop moiety, does not
show detectable coenzyme activity, but behaves as a
strong competitive inhibitor of AdoCbl [17]. Upon
incubation with apoenzyme in the presence of sub-
strate, this analog undergoes irreversible cleavage of its
Co–C bond, forming an enzyme-bound Co(II)-contain-
ing species. Adenosylcobinamide neither functions as a
coenzyme nor binds tightly to apoenzyme. It is thus
evident that the phosphate group of the coenzyme
nucleotide loop is essential for tight binding to the
apoenzyme and therefore for subsequent activation of
the Co–C bond and catalysis.
The X-ray structure of diol dehydratase showed that
the enzyme exists as a dimer of heterotrimers and
binds cobalamin in the ‘base-on’ mode, namely with a
5,6-dimethylbenzymidazole moiety coordinating to the
cobalt atom [18], as suggested by EPR studies [19,20].
The structure of the enzyme in complex with ade-
ninylpentylcobalamin (AdePeCbl), an inactive coen-
zyme analog, revealed the presence of the adenine-
binding pocket in the active site of the enzyme [21]
(Fig. 1A). The adenine ring of the bound AdePeCbl is

nearly parallel to the corrin ring and faces pyrrole
ring C. It is trapped in the pocket by several hydrogen
bonds with amino acid residues in the a-subunit
(Fig. 1B). The overall structure of the complex is
essentially the same as that of the enzyme–cyanocobal-
amin (CN-Cbl) complex, except that the orientation of
the side chain OH group of Sera224 is largely rotated
to form a hydrogen bond with N3 of the adenine moi-
ety in the enzyme–AdePeCbl complex. Sera224 is the
only residue whose side chain is hydrogen-bonded with
the adenine ring. This residue is conserved between
diol dehydratases [22–24] and glycerol dehydratases
[25–27]. Other residues also form hydrogen bonds with
6-NH
2
, N1, or N7, but through the main chain amide
groups [21]. In addition, the e-NH
2
group of Lysb135
forms a salt bridge with the phosphate group of cobal-
amin (Fig. 1C) [18].
In this article, we report the roles of adenine anchor-
ing and ion pairing at the AdoCbl-binding site in diol
dehydratase catalysis. To study the functions of
Sera224 and Lysb135 by site-directed mutagenesis, we
prepared several mutant enzymes, in which either
Sera224 or Lysb135 is mutated to other amino acids,
and investigated their catalytic properties by kinetic
and spectroscopic analyses. The mechanism-based
inactivation of a mutant enzyme during catalysis is

also reported here.
Results
Expression and purification of mutant diol
dehydratases
Mutant apoenzymes in which Sera224 or Lysb135 was
mutated to another amino acid were expressed in Esc-
herichia coli cells and purified to homogeneity by the
same procedure as that described for the wild-type
enzyme [28] – that is, by extraction from crude mem-
brane fractions with a buffer containing 1% Brij35.
Purified preparations of the mutant enzymes were ana-
lyzed by PAGE under denaturing and nondenaturing
conditions. Upon SDS ⁄ PAGE (Fig. 2A), three bands
corresponding to the a-, b- and c-subunits were
observed in each mutant enzyme. Upon nondenaturing
PAGE (Fig. 2B), all the mutants were electrophoresed
as a single band that corresponds to the (abc)
2
complex.
Catalytic activity and kinetic properties of
Sera224 mutant diol dehydratases
As shown in Table 1, k
cat
values of the Sa224A and
Sa224N mutants were 19% and 5%, respectively, of
that of the wild-type enzyme, but rapid inactivation
took place with the Sa 224A mutant (Fig. 3A). The
k
cat
⁄ k

inact
values, which show the average numbers of
catalytic turnovers before inactivation [29], indicated
that both Sera224 mutants were inactivated after
8000–15 000 turnovers on average, whereas the wild-
type enzyme underwent inactivation after 7.5 · 10
5
turnovers. The result obtained with the Sa224A
mutant suggests that the hydrogen bond donation
from the side chain OH group of Sera224 to N3 of the
adenine ring of AdoCbl is important for the continu-
ous progress of catalytic cycles. In the case of the
Sa224N mutant, the hydrogen bond might not be
formed, because the side chain –CH
2
CONH
2
group of
Asn in the Sa224N mutant is longer than the –CH
2
OH
group of Ser in the wild-type enzyme.
To examine the possibility that such differences
might affect the affinities of the enzyme for ligands,
K i. Ogura et al. Residues involved in coenzyme B
12
binding
FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS 6205
apparent K
m

values for the coenzyme AdoCbl and a
substrate 1,2-propanediol, as well as K
i
values for an
inhibitor, CN-Cbl, were determined with the mutants.
K
m
values for AdoCbl (Table 2) and 1,2-propanediol
(Table 1) increased markedly for the Sa224N mutant,
as compared with the wild-type enzyme, whereas K
i
for CN-Cbl was not so much affected. In contrast, K
m
for AdoCbl and K
i
for CN-Cbl decreased slightly for
the Sa224A mutant, although K
m
for 1,2-propanediol
did not change. Thus, it became clear that the steric
crowding or inappropriate hydrogen bonding induced
by the relatively bulky side chain of Asn in the
Sa224N mutant lowers significantly the affinity for
the coenzyme and substrate as well as catalytic effi-
ciency (k
cat
⁄ K
m
).
Catalytic activities and kinetic properties of

Lysb135 mutant diol dehydratases
Table 1 indicates that k
cat
values of the Kb135R,
Kb135A and Kb135Q mutants were 58–76% that of
the wild-type enzyme at saturating concentrations of
KK
PDO
PDO
S224
S224
K135
K135
A
BC
Fig. 1. The structure of the coenzyme-binding site in diol dehydratase. (A) Stereo drawing of the hydrogen bonding and the ion pairing
interactions of AdePeCbl with Sera224 (S224) and Lysb135 (K135), respectively. PDO represents (S)-1,2-propanediol in this drawing. Pink
and green colors indicate the a- and b-subunits, respectively, darkening continuously from the N-terminal to the C-terminal sides. (B) Resi-
dues interacting with the adenine moiety of AdePeCbl. (C) Residues interacting with the phosphodiester group in the nucleotide loop of
cobalamin.
Residues involved in coenzyme B
12
binding K i. Ogura et al.
6206 FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS
AdoCbl, and rapid inactivation during catalysis was
not observed with these mutants (Fig. 3B). In contrast,
k
cat
of the Kb135E mutant was only 2% that of the
wild-type enzyme, and the k

cat
⁄ k
inact
value indicated
that this mutant underwent inactivation after 5.4 · 10
4
turnovers on average. These results indicate that a
positive charge in the b135 residue is not absolutely
required for catalytic activity, but the introduction of
a negative charge there greatly lowers the activity. The
former suggests that the salt bridge between the phos-
phate group of the coenzyme nucleotide loop moiety
and the side chain of Lysb135 is not essential for activ-
ity. The latter would be probably due to the electro-
static repulsion between the negatively charged
phosphate group and –COO
)
in the side chain of the
b135 residue.
The effects of Lysb135 mutations on K
m
values for
AdoCbl and 1,2-propanediol as well as K
i
for CN-Cbl
are summarized in Tables 1 and 2. For the Kb135R
mutant, K
m
for AdoCbl and K
i

for CN-Cbl were
rather smaller than those of the wild-type enzyme. This
indicates that cobalamins are bound to this mutant
more tightly than to the wild-type enzyme, probably
because the salt bridge formation between the cobala-
min phosphate group and the guanidinium group of
Arg in the Kb135R mutant is appropriate. For the
Kb135A and Kb135Q mutants, which have a neutral
side chain at the b135 residue, K
m
values for AdoCbl
and 1,2-propanediol and K
i
for CN-Cbl increased sig-
nificantly. These results suggest that the affinities of
the enzyme for cobalamins and substrate are lowered,
probably due to the inability of these mutants to form
a salt bridge with the phosphate group of cobalamins.
In contrast, the Kb135E mutant, which has a negative
charge at the b135 residue, showed a K
m
for AdoCbl
and a K
i
for CN-Cbl that were larger than those of the
wild-type enzyme by two orders of magnitude,
although K
m
for 1,2-propanediol was comparable to
the values of the Kb135A and Kb135Q mutants. It

can therefore be concluded that the positive charge in
the b135 residue is not essential for catalysis and is
A
B
Fig. 2. PAGE analysis of the purified preparations of mutant diol de-
hydratases. (A) SDS ⁄ PAGE. (B) Nondenaturing PAGE. Samples were
electrophoresed on 11% (A) and 7% (B) polyacrylamide gels, and the
resulting gels were subjected to protein staining with Coomassie
Brilliant Blue R-250. Molecular mass markers, SDS-7 (Sigma-Aldrich,
St Louis, MO, USA). BPB, bromophenol blue; wt, wild-type enzyme.
The bands of the a-, b- and c-subunits are indicated on the right (A).
The position of the (abc)
2
complexes is indicated on the right (B).
Table 1. Kinetic parameters of mutant diol dehydratases, determined at 37 °C. The k
cat
values were determined by the alcohol dehydroge-
nase–NADH coupled method using 1,2-propanediol as substrate. The k
inact
values were calculated from a change in the slope of a tangent
to the time course curve of the reaction. The K
m
values were determined by the 3-methyl-2-benzothiazolinone hydrazone method. Aver-
age ± standard deviation (n = 3). The AdoCbl concentrations used were 15 l
M for the wild-type enzyme, Sa224A mutant and Kb135R
mutant, 45 l
M for the Sa224N mutant, 30 lM for the Kb135A mutant, 57 lM for the Kb135Q mutant, and 150 l M for the Kb135E mutant.
Enzyme
k
cat

,s
)1
(%)
K
m
for
1,2-propanediol (m
M)
k
cat
⁄ K
m
· 10
)6
(s
)1
ÆM
)1
)
k
inact
(min
)1
) k
cat
⁄ k
inact
· 10
)4
Wild-type 336 (100) 0.15 ± 0.02 2.2 0.027 75

Sa224A 64 (19) 0.15 ± 0.01 0.43 0.46 0.8
Sa224N 17 (5) 1.90 ± 0.01 0.009 0.070 1.5
Kb135R 254 (76) 0.12 ± 0.01 2.1 0.027 56
Kb135A 196 (58) 0.39 ± 0.02 0.50 0.059 20
Kb135Q 211 (63) 0.39 ± 0.01 0.54 0.068 19
Kb135E 7.7 (2) 0.40 ± 0.06 0.019 0.0085 5.4
K i. Ogura et al. Residues involved in coenzyme B
12
binding
FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS 6207
only moderately important for cobalamin binding, but
the introduction of a negative charge largely prevents
the enzyme–cobalamin interaction.
Spectral changes of AdoCbl upon incubation with
mutant enzymes in the presence of substrate
Figure 4 shows the spectral changes of AdoCbl upon
incubation with mutant apoenzymes in the presence of
1,2-propanediol. AdoCbl underwent a spectral change
to cob(II)alamin (B
12r
) upon incubation with the wild-
type or Sa224A apoenzyme for 5 min – that is, the
absorbance at 525 nm decreased and a new peak at
478 nm appeared (Fig. 4A,B). These spectra reflect the
steady-state concentrations of cob(II)alamin during
catalysis. With the wild-type enzyme, it was not so
much different from the spectrum obtained at 30 min
of incubation. In the case of the Sa224A mutant, how-
ever, the peak at 478 nm increased gradually upon
prolonged incubation, and the spectrum obtained at

30 min of incubation resembled the typical spectrum
of cob(II)alamin. As the Sa224A holoenzyme was
completely inactivated by 10 min of incubation, this
spectrum should be that of the completely inactivated
holoenzyme of this mutant. The cob(II)alamin-like spe-
cies in the inactivated Sa224A holoenzyme was stable
even under aerobic conditions, but underwent oxida-
tion to aquacobalamin (aqCbl) upon denaturation of
the complex with guanidine-HCl under acidic condi-
tions. The spectrum thus obtained no longer changed
upon photoillumination, suggesting that the Co–C
bond of the coenzyme had been completely and irre-
versibly cleaved upon incubation with the Sa224A
mutant for 30 min in the presence of substrate. In con-
trast, the spectral change of AdoCbl upon incubation
with the Sa224N apoenzyme was rather small even at
30 min of incubation (Fig. 4C). The spectrum observed
after denaturation of this mutant holoenzyme was sim-
ilar to that of free AdoCbl and changed to that of
aqCbl upon photoillumination. Relatively low activity
and a small k
inact
of the Sa224N mutant (Table 1)
would account for the lower steady-state concentration
of cob(II)alamin species and the slower rate of irre-
versible cleavage of the Co–C bond with this mutant.
When AdoCbl was incubated with the Kb135A
mutant in the presence of substrate, a similar spectral
change was observed within 5 min (Fig. 4D). However,
the peak at 478 nm then decreased gradually, and

the absorbance at 356 nm and  530 nm increased
with time of incubation. These absorption peaks
are characteristic of the diol dehydratase-bound
Fig. 3. Time courses of 1,2-propanediol dehydration by mutant diol
dehydratases. (A) Sera224 mutants. (B) Lysb135 mutants. The alco-
hol dehydrogenase–NADH coupled method was used. The reaction
mixture consisted of 60 ng of the wild-type or 600 ng of a mutant
apoenzyme, 0.1
M 1,2-propanediol, 50 lg of yeast alcohol dehydro-
genase, 0.2 m
M NADH, 0.04 M potassium phosphate buffer
(pH 8.0), and AdoCbl, in a total volume of 1.0 mL. The reaction
was started by adding AdoCbl at a concentration given in the
legend to Table 1. The absorbance changes (DA at 340 nm) per
60 ng of enzyme are shown here.
Table 2. Binding affinities of mutant diol dehydratases for AdoCbl
and CN-Cbl, determined at 37 °C. The k
cat
values were obtained as
described in the legend to Table 1. Apparent K
m
and K
i
values were
determined by the 3-methyl-2-benzothiazolinone hydrazone method,
followed by Lineweaver–Burk plots. Average ± standard deviation
(n = 3).
Enzyme
K
m

for
AdoCbl
(l
M)
k
cat
⁄ K
m
(AdoCbl) · 10
)8
(s
)1
ÆM
)1
)
K
i
for
CN-Cbl
(l
M)
Wild-type 0.94 ± 0.11 3.6 1.5 ± 0.1
Sa224A 0.36 ± 0.05 1.8 0.63 ± 0.00
Sa224N 3.4 ± 0.2 0.05 2.3 ± 0.1
Kb135R 0.25 ± 0.04 10.2 0.51 ± 0.08
Kb135A 2.2 ± 0.4 0.89 1.4 ± 0.2
Kb135Q 5.4 ± 0.6 0.39 4.4 ± 0.5
Kb135E 127 ± 11 0.00061 463 ± 41
Residues involved in coenzyme B
12

binding K i. Ogura et al.
6208 FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS
hydroxocobalamin (OH-Cbl) [30], suggesting that the
oxidation of cob(II)alamin accompanies the inactiva-
tion of the Kb135A holoenzyme during catalysis.
Upon denaturation of the complex after 30 min of
incubation, the spectrum of enzyme-bound OH-Cbl
was at least partly converted to a free aqCbl-like spec-
trum. When the mixture was then photoilluminated,
the spectrum underwent a further change to that of
free aqCbl. It is therefore likely that AdoCbl was
mostly converted to the enzyme-bound OH-Cbl by
30 min of incubation, but a fraction of the coenzyme
still remained as AdoCbl at this time.
EPR spectra obtained with the Sa224A mutant
diol dehydratase
When the wild-type holoenzyme was incubated with
1,2-propanediol at 4 °C for 1 min under anaerobic
conditions, the typical EPR spectrum of reacting holo-
enzyme was obtained (Fig. 5A). The characteristic
high-field doublet signal with a splitting 14.3 mT
was assigned to the 1,2-propanediol-1-yl radical
(substrate-derived radical) [31], and the low-field broad
signal to the low-spin Co(II) of cob(II)alamin. Such a
spectrum arises from weak coupling in the Co(II)–
organic radical pair [32–34]. The intensity of the dou-
blet signal decreased within 3 min of incubation at
25 °C, and a new small peak with a g-value of  2.1
appeared upon further incubation for 30 min. The
latter low-field signal might be due to the inactivated

holoenzyme being formed. In contrast, only a trace of
the typical doublet signal of reacting holoenzyme was
observed with the Sa224A mutant upon incubation
with substrate at 4 °C for 1 min (Fig. 5B). This indi-
cates that the steady-state concentration of an organic
radical intermediate is very low with the Sa224A
mutant, which is consistent with the low catalytic
activity of this mutant. Upon further incubation at
25 °C for 3 min, the trace of doublet signal disap-
peared, and new signals with g-values of 2.08 and
 2.2 appeared. Although the radical species giving
the g = 2.08 signal has not yet been identified, the
relative intensity of this signal increased with time of
incubation, and the signal became predominant after
Fig. 4. Spectral changes of AdoCbl upon aerobic incubation with mutant diol dehydratases in the presence of 1,2-propanediol. Apoenzyme
(5 nmol) of the wild-type (A), Sa224A mutant (B), Sa224N mutant (C) or Kb135A mutant (D) was incubated at 30 °C with 4.5 nmol of AdoCbl
in 0.01
M potassium phosphate buffer (pH 8.0) containing 1.3 M 1,2-propanediol and 1% Brij35, in a total volume of 1.0 mL. Spectra were
taken at 5 min (thick solid lines) and 30 min (thin solid lines) after the addition of AdoCbl. Enzymes were then denatured by adding 6
M gua-
nidine–HCl and 0.06
M citric acid. After incubation at 37 °C for 10 min, the mixture was neutralized to pH 8 by adding 200 lLof1M potas-
sium phosphate buffer (pH 8.0) and 40 lLof5
M KOH. After the spectral measurement (broken lines), the mixture was photoilluminated in
an ice-water bath for 10 min with a 300 W tungsten light bulb from a distance of 20 cm, and the spectra were taken again (dotted lines).
Spectra are corrected for dilution. The spectra of apoenzymes were subtracted from the spectra obtained.
K i. Ogura et al. Residues involved in coenzyme B
12
binding
FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS 6209

33 min. It might be due to the same radical species as
appeared with the wild-type enzyme upon prolonged
incubation. The signal with a g-value of  2.2 might
be assigned to Co(II) of cob(II)alamin. These results
indicate that the Sa224A mutant undergoes rapid and
irreversible inactivation during catalysis by the extinc-
tion of an organic radical intermediate through unde-
sirable side reaction(s).
Fate of the adenosyl group of AdoCbl in
inactivation of a mutant holoenzyme during
catalysis
To study the fate of the adenosyl group, the upper
axial ligand of AdoCbl, in the inactivation of the
Sa224A holoenzyme during catalysis, adenosyl group-
derived product(s) from AdoCbl were identified. After
30 min of incubation of the Sa224A holoenzyme with
1,2-propanediol, the inactivated holoenzyme was dena-
tured, and product(s) formed from the coenzyme were
extracted and analyzed by HPLC on a reversed-phase
column. The only nucleoside product derived from the
adenosyl group was identified as 5¢-deoxyadenosine.
The retention time of 5¢-deoxyadenosine was  8 min
under the conditions employed. The formation of
adenine, adenosine, 4¢,5¢-anhydroadenosine, 5¢,8-cyclic
adenosine or adenosine 5¢-aldehyde was not observed
at all. It is therefore evident that the inactivation of
this mutant enzyme during catalysis is a mechanism-
based one, because the hydrogen abstraction from sub-
strate by the coenzyme adenosyl radical takes place as
the initial event of catalysis. The amount of 5¢-deoxy-

adenosine formed from 4.6 nmol of the Sa224A
mutant and 15 nmol of AdoCbl was 4.7 nmol, which
corresponds to approximately one mol per mol of
enzyme. As diol dehydratase exists as a dimer of
heterotrimers, this result suggests that only one of the
two heterotrimeric units is involved in the formation
of 5¢-deoxyadenosine.
Recovery of active apoenzyme by resolution of
an inactivated mutant enzyme
A typical result of resolution experiments is shown in
Table 3. The Sa224A mutant was completely inacti-
vated by incubation with 1,2-propanediol for 30 min,
followed by dialysis. After resolution by acid ammo-
nium sulfate treatment, 56% of the original specific
activity of the mutant enzyme was recovered. The reso-
lution of cobalamin by this procedure was not com-
plete, and the resolved enzyme still contained OH-Cbl.
The cobalamin recovered in the supernatant was
aqCbl, and the extent of cobalamin resolution was
Fig. 5. EPR spectra observed upon incubation of the wild-type (A)
and Sa224A mutant (B) holodiol dehydratases with 1,2-propanediol.
The arrows correspond to g = 2.0. Holoenzymes were formed
under an argon atmosphere by incubating 1.9 mg (9.2 nmol) of sub-
strate-free wild-type and Sa224A apoenzymes at 25 °C for 3 min
with 50 nmol of AdoCbl in 0.65 mL of 0.05
M potassium phosphate
buffer (pH 8.0) containing 18 m
M sucrose monocaprate. The
enzyme reaction was started by adding 50 lmol of 1,2-propanediol
in 0.05 mL. After 1 min at 4 °C, the reaction mixture was rapidly

frozen in an isopentane bath that had been previously cooled to
approximately )160 °C, and then in a liquid nitrogen bath. EPR
spectra were taken at )130 °C. After the first measurement, the
mixture was incubated at 25 °C for 3 min and frozen again, as
described above, for the second measurement. The mixture was
then incubated at 25 °C for an additional 5 min and 25 min for the
third and fourth measurements.
Residues involved in coenzyme B
12
binding K i. Ogura et al.
6210 FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS
estimated to be 61% from the cobalamin contents of
the 1,2-propanediol-inactivated Sa224A holoenzyme
and of the resolved enzyme. This value of cobalamin
resolution is in good agreement with the recovery of
enzyme activity. It was therefore concluded that
resolved apoenzyme recovered from the inactivated
mutant holoenzyme could be reconstitutable to fully
active holoenzyme – that is, the Sa224A apoenzyme
itself did not undergo damage in the mechanism-based
inactivation by the substrate 1,2-propanediol.
Inactivation of mutant holoenzymes by O
2
in the
absence of substrate
The holoenzyme of diol dehydratase undergoes irre-
versible inactivation by O
2
in the absence of substrate
[35]. This inactivation is accompanied by the irrevers-

ible Co–C bond cleavage of the enzyme-bound coen-
zyme, forming OH-Cbl. It is thus believed that the
inactivation is caused by the reaction of the activated
Co–C bond with O
2
, and thus reflects the extent of
Co–C bond activation upon the coenzyme binding to
apoenzyme in the absence of substrate. As shown in
Table 4, the inactivation followed pseudo-first-order
reaction kinetics, with a rate constant (k
inact,O2
)of
0.20 min
)1
for the wild-type holoenzyme. To examine
the contributions of enzyme–coenzyme interactions at
the Sera224 and Lysb135 residues to Co–C bond acti-
vation, rates of O
2
inactivation of mutant holoenzymes
in the absence of substrate were determined. The rate
constants of O
2
inactivation for the Lysb135 mutants
were in the range 0.13–0.17 min
)1
, which is slightly
slower but almost comparable to that of the wild-type
holoenzyme. In contrast, the rate of O
2

inactivation of
mutant holoenzymes was very slow for the Sa224A
mutant, and inactivation was not observed with the
Sa224N mutant. These results suggest that the appro-
priate hydrogen bonding between the side chain OH
group of Sera224 and N3 of the adenine ring of the
coenzyme adenosyl group is important for activation
of the Co–C bond in the absence of substrate as well.
On the other hand, ion pairing between the e-NH
2
group of Lysb135 and the phosphate group of the
coenzyme nucleotide loop seems not to be essential for
Co–C bond activation. This would be reasonable,
because the cobalamin moiety of AdoCbl is accommo-
dated to the cobalamin-binding site of the enzyme
through multiple interactions with many amino acids.
Discussion
The homolytic fission of the Co–C bond of enzyme-
bound AdoCbl leads to the introduction of an adenosyl
radical, a catalytic radical, into the active sites. This is
an essential early event in all of the AdoCbl-dependent
enzymatic reactions [1–3,7,8]. We synthesized various
coenzyme analogs in which one of the structural compo-
nents is substituted by a closely related group, and used
them as probes to investigate the mechanism of enzy-
matic activation (labilization) of the coenzyme Co–C
bond as well as the role of each structural component of
the coenzyme in the interaction with diol dehydratase
[1,11–17]. It was demonstrated that the cobalamin moi-
ety [14–17,29] and the adenosyl group [11–13] are

required for its tight binding to the apoenzyme and for
activation of the Co–C bond, respectively, and that the
‘adenine-attracting effect’ of the apoenzyme is a major
element that weakens the Co–C bond [36,37]. Later, the
X-ray structures of the diol dehydratase–AdePeCbl
complex revealed that the enzyme has a cobalamin-bind-
ing site [18] and an adenine-binding pocket [21] for Ado-
Cbl. A modeling study using X-ray structures suggested
that the tight binding of AdoCbl to both of these sites
induces marked distortions, including both angular
strains and tensile force, that inevitably lead to Co–C
bond cleavage [21,38]. We proposed this ‘steric strain
model’ as the molecular mechanism for the enzymatic
activation of the coenzyme’s Co–C bond.
The X-ray structures of diol dehydratase show that
the coenzyme adenine moiety is anchored in the pro-
tein by hydrogen bonding of N3 with the side chain
OH group of Sera224, of 6-NH
2
and N7 with main
chain amide groups of other residues, and of N1 with
a water molecule [21]. Cobalamin is accommodated to
a space that is mainly surrounded by hydrophilic
groups [18]. Five amide groups out of six peripheral
Table 3. Resolution of 1,2-propanediol-inactivated Sa2424A mutant
holoenzyme by acid ammonium sulfate treatment.
Specific activity,
UnitsÆmg
)1
(%)

B
12
bound,
l
M(%)
Apoenzyme used 2.5 (100) 0.0 (0)
Inactivated holoenzyme 0.0 (0) 3.1 (100)
Resolved enzyme 1.4 (56) 1.2 (39)
Table 4. O
2
inactivation of mutant holo-diol dehydratases in the
absence of substrate, determined at 37 °C.
Enzyme k
inact,O2
(min
)1
)
Wild-type 0.20
Sa224A 0.03
Sa224N Not inactivated
a
Kb135R 0.17
Kb135A 0.16
Kb135Q 0.13
a
No inactivation was observed for at least 10 min.
K i. Ogura et al. Residues involved in coenzyme B
12
binding
FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS 6211

side chains of the corrin ring form hydrogen bonds
with five amino acids in the a-subunit and three in the
b-subunit. The phosphate group of cobalamin forms a
salt bridge between the e-amino group of Lysb135, in
addition to hydrogen bonds with the two residues in
the b-subunit. In this study, the impacts of adenine
anchoring and ion pairing on catalysis were evaluated
by site-directed mutagenesis at Sera224 and Lysb135.
The Sa224A mutant, which cannot form a hydrogen
bond with N3 of the adenine moiety, showed a relative
activity (k
cat
) of 19% and decreased sensitivity of the
holoenzyme to O
2
in the absence of substrate, suggest-
ing the importance of hydrogen bonding between N3
of the adenine moiety and the side chain OH group of
Sera224 for activation of the coenzyme Co–C bond.
On the other hand, the Sa224N mutant had only 5%
relative activity, which was much lower than that of
the Sa224A mutant. Its complex with AdoCbl did not
show the sensitivity to O
2
in the absence of substrate.
One possibility is that the hydrogen bond might not be
formed or be formed at an improper position with the
Sa224N mutant. The other possibility is that it might
be due to the coenzyme-binding problems caused by
the Sa224N mutation, regardless of the hydrogen

bonding interaction with N3.
The O
2
inactivation of the holoenzyme occurs only in
the absence of substrate. The inactivation mechanism
remains unclear at present, and the identification of the
inactivation products would provide important clues to
solve this problem. It was well established from the spec-
tral changes that OH-Cbl is formed upon the inactiva-
tion [35]. However, products from the adenosyl group of
AdoCbl have not yet been definitely identified [35]
(M. Yamanishi, S. Yamanaka & T. Toraya, unpub-
lished results). Product(s) from O
2
also remain to be
identified. O
2
inactivation can be considered to be clo-
sely related to catalysis, because only the complexes with
active coenzyme analogs undergo this inactivation,
except for the complexes with 3-deazaAdoCbl [12], neb-
ularylcobalamin (deamino analog of AdoCbl) [11], and
aristeromycylcobalamin (carbocyclic analog of AdoCbl)
[11,39]. The substrate facilitates Co–C bond cleavage by
inducing conformational changes that increase the steric
strain of the Co–C bond of enzyme-bound coenzyme
[38]. Therefore, these results suggest that the Co–C bond
activation in the absence of substrate is not sufficient
with coenzyme analogs that lack hydrogen bonding
interactions with the enzyme.

The k
cat
⁄ k
inact
value is a good measure of the resis-
tance of holoenzymes to mechanism-based inactivation.
These ratios for the Sa224A and Sa224N mutants indi-
cate that these enzymes underwent inactivation after
only 8000 and 15 000 turnovers, respectively, on aver-
age. We have previously reported that the wild-type
enzyme shows a small k
cat
⁄ k
inact
value when 3-dea-
zaAdoCbl, a coenzyme analog that cannot form a
hydrogen bond with Sera224, is used as coenzyme [12].
It can thus be concluded that the proper hydrogen
bonding between N3 of the adenine moiety and Sera224
plays an essential role in protecting highly reactive radi-
cal intermediate(s) from undesired side reactions, proba-
bly through stable anchoring of the adenine moiety to
the adenine-binding pocket. Upon the inactivation of
Sa224A, a cob(II)alamin-like spectrum was observed.
The EPR spectrum suggested that cob(II)alamin and an
unidentified radical species were formed from AdoCbl
and accumulated upon prolonged incubation with the
Sa224A mutant. A stoichiometric amount of 5¢-deoxy-
adenosine was formed upon inactivation from the
enzyme-bound coenzyme, but the apoenzyme itself was

not damaged. Thus, the inactivation of the Sa224A
mutant during catalysis was concluded to be a mecha-
nism-based one, as shown below:
AdoH
AdoH
E
Ado
Cbl
III
SH
E
Ado
Cbl
II
SH
•
•
E
Cbl
II
•S
•
side
reaction
E
Cbl
II
•
where E is enzyme, SH is substrate, Cbl is cobalamin,
and Ado is 5¢-deoxy-5¢-adenosyl.

Mutant enzymes in which Lysb135 was substituted
with Arg, Ala or Gln showed relatively high activity
and relatively large k
cat
⁄ k
inact
values, whereas the
Kb135E mutant possessed a trace of activity and a
small k
cat
⁄ k
inact
value. Binding affinities for AdoCbl
and CN-Cbl were strengthened when Lysb135 was
substituted with Arg, being almost the same as the
those of the wild-type enzyme upon the Ala substitu-
tion, and slightly lowered upon the Gln substitution at
the b135 residue. This might be due to hydrogen bond-
ing and interactions other than the ion pairing being
strong enough to maintain the tight binding of cobala-
min. In contrast, the Kb135E mutant showed more
than 100-fold lower affinity for both cobalamins.
These results indicate that a salt bridge between the
phosphate group and the side chain of the b135 resi-
due is not essential for either catalysis or cobalamin
binding, but the introduction of a negative charge in
the b135 residue destroyed the affinity for cobalamins
and lowered the enzyme activity to 2% even when
AdoCbl was used at a concentration higher than its
K

m
. It was also evident from the decreased k
cat
⁄ k
inact
value with the Kb135E mutant that the electrostatic
repulsion between the cobalamin phosphate group and
the side chain –COO
)
of Glub135 also results in the
destabilization of the reactive radical intermediate(s)
during catalysis.
Residues involved in coenzyme B
12
binding K i. Ogura et al.
6212 FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS
Experimental procedures
Materials
Crystalline AdoCbl was a gift from Eisai Co. Ltd (Tokyo,
Japan). Crystalline CN-Cbl was obtained from Glaxo
Research Ltd (Greenford, UK). Other chemicals were analyti-
cal grade reagents and were used without further purification.
Construction of expression plasmids for mutant
diol dehydratases
The mutations described in this article were introduced into
the diol dehydratase genes (pddABC)ofKlebsiella oxytoca
(formerly Aerobacter aerogenes) ATCC8724, using a Quik-
Change site-directed mutagenesis kit (Stratagene, La Jolla,
CA, USA). pUSI2E(DD) [22], an expression plasmid for the
wild-type enzyme, was used as a template. The mutagenic

sense primers used were 5¢-ctacgccgaaaccatc
gccgtctacggcac-3¢
for Sa224A, 5¢-ctacgccgaaaccatc
aacgtctacggcac-3¢ for
Sa224N, 5¢-ggcatccagtcg
agaggcaccacggtgatc-3¢ for Kb135R,
5¢-ggcatccagtcg
gcaggcaccacggtgatc-3¢ for Kb135A, 5¢-ggcatc
cagtcg
caaggcaccacggtgatc-3¢ for Kb135Q, and 5¢-gcatc
cagtcg
gaaggcaccacggtgatc-3¢ for Kb135E, in which underlin-
ing indicates amino acid substitutions. The oligonucleotides
having the complementary sequences in the opposite direc-
tion were used as the respective antisense primers. It was con-
firmed by sequencing of the DNA region encompassing the
entire diol dehydratase genes and tac promoter that no unin-
tended mutations had been incorporated during mutagenesis.
Expression and purification of mutant diol
dehydratases
E. coli XL1-Blue cells were transformed with the above-men-
tioned expression plasmids. Recombinant E. coli cells were
grown aerobically in LB medium containing 0.1% 1,2-pro-
panediol and ampicillin (50 lgÆmL
)1
), and induced by 1 mm
isopropyl b-d-1-thiogalactopyranoside, as described previ-
ously [22]. Mutant apoenzymes were purified from over-
expressing E. coli cells, essentially as described previously for
the recombinant wild-type enzyme [28]. The DEAE–cellulose

chromatography step was omitted, as the enzymes extracted
from crude membrane fractions were found to be almost
homogeneous. However, the DEAE–cellulose-purified
apoenzymes were used in the EPR experiments.
Substrate-free apoenzymes
Substrate-free apoenzymes for the measurements of K
m
values for 1,2-propanediol and rates of inactivation of
holoenzymes in the absence of substrate were obtained by
dialysis at 4 °C for 36 h against 100 volumes of 50 mm
potassium phosphate buffer (pH 8.0) containing 0.1%
Brij35 with two buffer changes.
Enzyme and protein assays
Diol dehydratase activity was routinely measured by the
3-methyl-2-benzothiazolinone hydrazone method, using
1,2-propanediol as substrate [11]. One unit is defined as the
amount of enzyme activity that catalyzes the formation of
1 lmol of propionaldehyde ⁄ min at 37 °C under the stan-
dard assay conditions. Time courses of the diol dehydratase
reaction were measured by the alcohol dehydrogenase–
NADH coupled method [29].
The protein concentration of purified enzyme was deter-
mined by measuring the absorbance at 280 nm. The molar
absorption coefficient at 280 nm, calculated by the method
of Gill & von Hippel [40], for diol dehydratase is
120 500 m
)1
Æcm
)1
[41].

PAGE
PAGE analyses of purified mutant enzymes were performed
under nondenaturing conditions as described by Davis [42],
in the presence of 0.1 m 1,2-propanediol [22], and under
denaturing conditions as described by Laemmli [43]. Pro-
teins were stained with Coomassie Brilliant Blue R-250.
EPR measurements
The wild-type and the Sa224A mutant apoenzymes purified
as described previously [28] were used. Substrate-free apoen-
zyme solution [1.9 mg of protein in 0.6 mL of 50 mm potas-
sium phosphate buffer (pH 8.0) containing 20 mm sucrose
monocaprate] was mixed at 0 °C with AdoCbl solution
(50 nmol in 0.05 mL) in a quartz EPR tube (outside diameter
5 mm) stoppered with a rubber septum. After replacement of
the air in the tube with argon by repeated evacuation and
flushing with argon three times, holoenzymes were formed,
reacted with 1,2-propanediol, and rapidly frozen as described
in the legend to Fig. 5. The frozen sample was transferred to
the EPR cavity and cooled with a cold nitrogen gas flow con-
trolled by a Eurotherm B-VT 2000 temperature controller.
EPR spectra were taken as described previously [44,45] at
)130 °C on a Bruker ESP-380E spectrometer modified with
a Gunn diode X-band microwave unit. EPR microwave
frequency was 9.484–9.488 GHz, modulation amplitude was
1 mT, modulation frequency was 100 kHz, and microwave
power was 10 mW.
Fate of the adenosyl group of AdoCbl in inactivation
of mutant holoenzymes during catalysis
The adenosyl group-derived product(s) formed from AdoCbl
in the inactivation of a mutant holoenzyme during catalysis

was identified as described previously [46]. Substrate-free
apoenzyme (1.0 mg, 4.6 nmol) was incubated at 37 °C for
30 min in the dark with 15 lm AdoCbl in the presence
of 0.1 m 1,2-propanediol. The enzyme protein was then
K i. Ogura et al. Residues involved in coenzyme B
12
binding
FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS 6213
denatured by adding ethanol to a final concentration of
80%. The mixture was heated at 90 °C for 10 min, and then
centrifuged at 8060 g for 10 min. The supernatant was evap-
orated to a small volume and taken up into 0.5 mL 15%
methanol containing 1% acetic acid. The nucleoside product
from the adenosyl group was analyzed by HPLC using a
Cosmosil C
18
column (0.46 · 15 cm) (Nacalai Tesque,
Kyoto, Japan) with 15% methanol containing 1% acetic
acid as a mobile phase. The amount of the product formed
was determined from its peak height upon development of
the calibrated column at a flow rate of 0.4 mLÆmin
)1
.
Resolution of inactivated mutant holoenzyme by
acid ammonium treatment
The enzyme purified as described above was not appropriate
for the resolution experiment, because Brij35 was salted out
in the process of acid ammonium sulfate fractionation and
was no longer effective for solubilizing the enzyme. Instead,
trypsin-solubilized Sa224A apoenzyme was prepared as

described previously [46] and successfully used in the resolu-
tion experiments. The Sa224A holoenzyme inactivated dur-
ing catalysis was obtained by the incubation of trypsin-
solubilized apoenzyme (1.5 mg) at 37 °C for 30 min with
AdoCbl with the substrate 1,2-propanediol, followed by
dialysis, and the inactivated holoenzyme obtained was then
resolved by acid ammonium sulfate treatment, as described
previously [47]. The activities of the apoenzyme used, the
inactivated holoenzyme and the resolved enzyme were mea-
sured by the alcohol dehydrogenase–NADH coupled
method in the presence of added AdoCbl. The amounts of
cobalamin bound to the inactivated holoenzyme and the
resolved enzyme were determined spectrophotometrically.
O
2
inactivation of holoenzymes in the absence
of substrate
Appropriate amounts of substrate-free wild-type and
mutant apoenzymes were incubated aerobically with 72 lm
AdoCbl at 37 °C for various time periods in 35 mm potas-
sium phosphate buffer (pH 8.0) containing 50 mm KCl.
Substrate (0.1 m 1,2-propanediol) was then added to stop
the O
2
inactivation, and the remaining enzyme activities
were measured by incubating the mixtures at 37 °C for an
additional 10 min.
Acknowledgements
This work was supported in part by Grants-in-Aid for
Scientific Research [(B) 13480195 and 17370038 and

Priority Areas 753 to T. Toraya, and (C) 14580627 to
T. Tobimatsu] 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 to T. Toraya.
We thank Y. Kurimoto for her assistance in manu-
script preparation.
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