 |
INTRODUCTION |
Diol dehydratase (propane-1,2-diol hydro-lyase, EC 4.2.1.28) is an
enzyme that catalyzes the adenosylcobalamin
(AdoCbl)1
-dependent conversion of 1,2-propanediol, glycerol, and
1,2-ethanediol to the corresponding aldehydes (1, 2). This enzyme is
inducibly formed by some genera of Enterobacteriaceae, such
as Klebsiella and Citrobacter, and other bacteria
when they are grown anaerobically in medium containing 1,2-propanediol
(3, 4). When some of these bacteria are grown anaerobically on
glycerol, they form glycerol dehydratase as well (3, 4). Diol and
glycerol dehydratases are important for producing essential electron
acceptors in the fermentation of 1,2-propanediol and glycerol,
respectively (5-9). Klebsiella oxytoca ATCC 8724 is
defective in glycerol dehydratase (10, 11), but still capable of
fermenting glycerol. This is because a low level of diol dehydratase
induced by glycerol substitutes for isofunctional glycerol dehydratase
(2, 7, 12, 13).
Diol dehydratase as well as glycerol dehydratase undergoes concomitant,
irreversible inactivation by glycerol during catalysis (2, 14-16).
This inactivation is mechanism-based and involves irreversible cleavage
of the Co
C bond of AdoCbl forming 5'-deoxyadenosine and a modified
coenzyme (4, 14). Irreversible inactivation of the enzyme results from
the tight binding of the modified, inactive cobalamin (4, 14, 17). Such
suicide inactivation seemed enigmatic because glycerol is a growth
substrate for K. oxytoca and Klebsiella
pneumoniae. This apparent inconsistency was solved by our finding
that the glycerol-inactivated enzymes in permeabilized cells (in
situ) of K. oxytoca and K. pneumoniae undergo rapid reactivation in the presence of free AdoCbl, ATP, and
Mg2+ (or Mn2+) (13, 18). Because the
reactivation was detectable only in situ but not in
vitro, it remained unclear whether the reactivation is caused by a
specific proteinous factor. Recently, we have identified the two open
reading frames located in the 3'-flanking of the K. oxytoca
diol dehydratase genes as the genes encoding a reactivating factor for
glycerol-inactivated diol dehydratase and designated them as
ddrA and ddrB genes (19, 20).
This article reports biochemical demonstration that purified DdrA and
DdrB proteins form a tight complex that serves as a reactivating
factor for glycerol-inactivated holoenzyme as well as
O2-inactivated holoenzyme in vitro in the
presence of free AdoCbl, ATP, and Mg2+. Evidence for the
reactivating factor-mediated exchange of an enzyme-bound,
adenine-lacking cobalamins for a free, adenine-containing cobalamin is
also described here.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Crystalline CN-Cbl was obtained from Glaxo
Research Laboratories, Ltd., Greenford, UK. AdoCbl was a gift from
Eisai Co., Ltd., Tokyo, Japan. AdePeCbl was synthesized by the
published procedure (21). Recombinant diol dehydratase was purified to
homogeneity from an overexpressing Escherichia coli JM109
harboring expression plasmid pUSI2E(DD) (22).
Construction of Expression Plasmids--
We have constructed
expression plasmid pCXV(6/5b) for the ddrAB genes using
vector pCXV (20). Because copy numbers of plasmids containing
replication origin of p15A, like pCXV are less than those of plasmids
containing replication origin of pBR322, like pUSI2E (23), (10-12 and
15-20 copies/cell, respectively (24)), we transferred the genes
from pCXV(6/5b) to another expression vector, pUSI2ENd. The
6.6-kilobase HindIII-BglII fragment
containing the tac promoter sequence and the ddrA
and ddrB genes was excised from pCXV(6/5b) and ligated to
the 5-kilobase HindIII-BglII fragment of
pUSI2ENd(DD) to construct pUSI2ENd(6/5b). pUSI2ENd(DD) was a derivative
of pUSI2E(DD) (23), an expression plasmid for the diol dehydratase
genes. The unnecessary NdeI site on the vector region of
pUSI2E(DD) was eliminated in pUSI2ENd(DD). E. coli JM109 was
used as a host for expression plasmid pUSI2ENd(6/5b).
Cultivation of Overexpressing E. coli Strain--
Recombinant
DdrA and DdrB proteins of K. oxytoca were purified to
homogeneity from E. coli JM109 harboring expression plasmid pUSI2ENd(6/5b) that was aerobically grown at 30 °C in LB medium containing ampicillin (50 µg/ml).
Isopropyl-1-thio-
-D-galactopyranoside was added to a
concentration of 1 mM for induction, and cells were
harvested in the late logarithmic phase.
Protein Assays--
During purification of diol dehydratase and
the ddrA and ddrB gene products, protein
concentrations were routinely estimated by the method of Lowry et
al. (25) with crystalline bovine serum albumin as a standard. The
concentration of purified diol dehydratase was determined by measuring
the absorbance at 280 nm. The molar absorption coefficient at 280 nm
(
M, 280) calculated by the method of Gill and von
Hippel (26) for diol dehydratase from its deduced amino acid
compositions (23) and subunit structure (22) was 120,500 M
1 cm
1. Based on the molecular
weight predicted,
1%, 280 was calculated to be 5.81 for diol dehydratase.
PAGE--
PAGE was performed under non-denaturing conditions
essentially as described by Davis (27), except that 5 mM dithiothreitol was added in the gel, or under denaturing
conditions as described by Laemmli (28). Protein was stained with
Coomassie Brilliant Blue R-250. Densitometric analysis of gels was
performed with a Printgraph AE-6911CX system (ATTO, Tokyo, Japan) and
the NIH-Image program, Version 1.61 (National Institutes of Health).
Edman Sequencing of the Subunits--
A purified preparation of
the complex of the DdrA and DdrB proteins was separated into the
subunits (A and B polypeptides, respectively) by SDS-PAGE (15.0%) and
electrophoretically transferred to a polyvinylidene difluoride membrane
(Applied Biosystems). Protein bands were visualized with Coomassie
Brilliant Blue R-250, excised, and analyzed for
NH2-terminal amino acid sequences on an Applied Biosystems
491 protein sequencer.
Molecular Weight Determination by Gel Filtration--
The
molecular weight of the complex (putative reactivating factor) was
determined by gel filtration on Superose 6 column (HR10/30) using a
FPLC system (Amersham Pharmacia Biotech). The purified factor and
molecular weight marker proteins were applied to the column, which was
equilibrated with 50 mM potassium phosphate buffer (pH 8.0)
containing 0.1 M KCl and developed with the same buffer at
a flow rate of 0.4 ml/min. The elution of proteins was monitored by
A280.
Enzyme Assays--
The amount of aldehydic products formed by
diol dehydratase reaction was determined by the
3-methyl-2-benzothiazolinone hydrazone method (29). One unit of diol
dehydratase is defined as the amount of enzyme activity that catalyzes
the formation of 1 µmol of propionaldehyde/min at 37 °C.
Reactivation of the inactivated holoenzymes and activation of the
enzyme·CN-Cbl complex by the reactivating factor was determined using
1,2-propanediol or glycerol as a substrate in the presence of 21 µM AdoCbl, 24 mM ATP, and 24 mM
MgCl2.
The capability of reactivating factor to reactivate
glycerol-inactivated or O2-inactivated holodiol dehydratase
was assayed in vitro using 1,2-propanediol as substrate. The
capability of it to activate the inactive enzyme·CN-Cbl complex was
also assayed in vitro because these two capabilities
in situ were shown to be well correlated (18).
 |
RESULTS |
Purification of Recombinant DdrA and DdrB Proteins--
The
ddrA and ddrB gene products were co-purified from
extracts of overexpressing E. coli. All operations were
performed at 0-4 °C. Throughout the purification steps, purity of
the proteins in each fraction was analyzed by SDS-PAGE.
About 10 g of wet cells grown at 30 °C were suspended in 50 ml
of 50 mM potassium phosphate buffer (pH 8.0) containing 2 mM EDTA and 2 mM PMSF and disrupted by
sonication for 10 min at 240 W with a Kaijo Corp. TA-5287 ultrasonic
destruction system (Japan). After centrifugation at 27,200 × g for 30 min, the supernatant was collected. The precipitate
was washed with 60 ml of the same buffer, and the washing was combined
with the supernatant (cell-free extract).
Solid ammonium sulfate was added to the cell-free extract to 15%
saturation. After centrifugation, solid ammonium sulfate was added
again to the supernatant to 35% saturation. The precipitate was
dissolved in 20 ml of 50 mM potassium phosphate buffer (pH 8.0) containing 2 mM EDTA and 2 mM PMSF and
dialyzed for 1 day against 2 liters of 5 mM potassium
phosphate buffer (pH 8.0) containing 0.5 mM EDTA with one
buffer change.
The dialysate was applied to a DEAE-cellulose column (bed volume, 100 ml) that was equilibrated with 10 mM potassium phosphate buffer (pH 8.0). The column was washed successively with 500 ml of the
same buffer and with 600 ml of 5 mM potassium phosphate buffer (pH 8.0) containing 130 mM KCl and then developed
with 800 ml of 5 mM potassium phosphate buffer (pH 8.0)
containing 200 mM KCl and 200 ml of the same buffer
containing 250 mM KCl. The DdrA and DdrB
proteins-containing fractions were pooled. The buffer concentration was
lowered by repeating concentration by ultrafiltration through a Diaflo
PM-10 membrane (Amicon) and dilution in 1 mM potassium
phosphate buffer (pH 8.0).
The resulting solution was applied to a hydroxyapatite column (bed
volume, 70 ml) which was equilibrated with 2 mM potassium phosphate buffer (pH 8.0). The column was washed successively with 280 ml of the same buffer and with 200 ml of 6 mM potassium phosphate buffer (pH 8.0) and then developed with 350 ml of 10 mM potassium phosphate buffer (pH 8.0). The DdrA and DdrB
proteins-containing fractions were pooled and concentrated to about 1.7 ml by ultrafiltration through a Diaflo PM-10 membrane and Centriplus (Amicon).
The concentrated solution was loaded onto a Sephadex G-200 column (bed
volume, 150 ml) which was equilibrated with the 50 mM
potassium phosphate buffer (pH 8.0). The column was developed with the
same buffer, and the fractions containing the DdrA and DdrB proteins
were pooled, concentrated to about 5 ml by ultrafiltration through a
Centriplus, and stored at
80 °C.
Purity, Molecular Weight, and Subunit Structure of the Purified
Reactivating Factor--
It is evident that the two bands with
Mr of 64,000 and 14,000 (designated A and B
polypeptides, respectively) were overexpressed in E. coli
JM109 carrying pUSI2ENd(6/5b) (Fig.
1A). Both of these bands were
progressively enriched upon purification. When the purified preparation
was electrophoresed under non-denaturing conditions in the presence of
dithiothreitol, it migrated as a single band (Fig. 1B).
Therefore, it was clear that the two polypeptides were co-purified as a
tight complex. Because the predicted molecular weights of the DdrA and
DdrB proteins are 64,266 and 13,620, respectively (20), it was
highly suggested that the A and B polypeptides are the products of
these genes. The NH2-terminal 10-amino acid sequences of
the A and B polypeptides determined by Edman sequencing were MRYIAGIDIG
and MNGNHSAPAI, respectively. These sequences agreed completely with
those deduced from the nucleotide sequences of the ddrA and
ddrB genes (20). Thus, the A and B polypeptides were
undoubtedly identified as the ddrA and ddrB gene
products, respectively. These results indicate that the DdrA and DdrB
proteins are co-purified and exist as a tight complex under
non-denaturing conditions. This complex was considered as a putative
reactivating factor.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 1.
Purification of the DdrA and DdrB
proteins. Fractions at each purification step were subjected
to SDS-PAGE (denaturing) on 12.5% polyacrylamide gel (A)
and PAGE (non-denaturing) on 6.5% polyacrylamide gel containing 5 mM dithiothreitol (B). Protein staining of the
resulting gels were carried out as described under "Experimental
Procedures." Molecular weight markers were SDS-7 (Sigma)
(A). Positions of the A and B polypeptide are indicated with
arrowheads to the right of the gel
(A).
|
|
To determine the subunit composition of the factor, the complex
purified to homogeneity was separated into subunits by SDS-PAGE in a
15% gel and stained with Coomassie Brilliant Blue R-250. Densitometric
analysis of the bands, together with molecular weights of the subunits
predicted from the amino acid composition, indicated that the molar
ratio of the A and B polypeptides in the complex was approximately
1:1.1. The apparent molecular weight of the complex determined by FPLC
with a calibrated Superose 6 column (HR 10/30) was approximately
150,000 (data not shown). By taking the predicted molecular weights of
the subunits into consideration, it can be concluded that the subunit
structure of the putative reactivating factor is most likely
A2B2.
The molar absorption coefficient at 280 nm (
M, 280) for
the complex (reactivating factor), calculated by the method of Gill and
von Hippel (26) from its amino acid compositions and subunit structure,
was 58,140 M
1 cm
1.
1%, 280 for the reactivating factor was calculated to
be 3.73 on the basis of the predicted molecular weight.
In Vitro Reactivation of Inactivated Holoenzymes by the
Reactivating Factor--
The capability of the putative reactivating
factor to reactivate the glycerol-inactivated holodiol dehydratase was
examined in vitro using 1,2-propanediol as substrate. As
illustrated in Fig. 2A,
in vitro reactivation of the glycerol-inactivated holoenzyme by the purified factor in the presence of AdoCbl, ATP, and
Mg2+ was observed for the first time. The reactivation did
not take place at all without the factor or with the factor but in the absence of ATP/Mg2+. Free AdoCbl was also absolutely
required for the reactivation (data not shown). Thus, it is evident
that the factor actually functions as a reactivating factor for
glycerol-inactivated diol dehydratase. The product formed increased
exponentially at the initial stage of reaction and then almost linearly
with time of incubation. By comparison of the maximum slope with the
control, the extent of reactivation under the conditions employed was
estimated to be approximately 64%.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Reactivation of glycerol-inactivated
(A) and O2-inactivated
(B) holodiol dehydratases by the reactivating
factor. Glycerol-inactivated holoenzyme was prepared by incubation
of apoenzyme (331 units) with 50 µM AdoCbl at 37 °C
for 30 min in 2.5 ml of 0.05 M potassium phosphate buffer
(pH 8) containing 30% glycerol, followed by dialysis at 4 °C for
43 h against 800 volumes of 0.05 M potassium phosphate
buffer (pH 8) containing 2% 1,2-propanediol.
O2-inactivated holoenzyme was prepared by incubation of
substrate-free apoenzyme (12.6 units) with 49 µM AdoCbl
at 37 °C for 30 min in 0.18 ml of 0.05 M potassium
phosphate buffer (pH 8). Glycerol-inactivated holoenzyme (1.5 units) or
O2-inactivated holoenzyme (1.5 units) was incubated at
37 °C for the indicated time periods with ( , ) and without
( , ) 47 µg of the reactivating factor in 0.02 M
potassium phosphate buffer (pH 8) containing 21 µM AdoCbl
and 1.2 M 1,2-propanediol in the presence ( , ) and
absence ( , ) of 24 mM ATP/24 mM
MgCl2, in a total volume of 50 µl. Time course of
1,2-propanediol dehydration with non-inactivated enzyme was measured as
a control ( ). The reaction was terminated by adding 50 µl of 0.1 M potassium citrate buffer (pH 3.6). After removal of
precipitate by centrifugation, the reaction mixture was appropriately
diluted for determining the amount of propionaldehyde formed by the
3-methyl-2-benzothiazolinone hydrazone method (18).
|
|
Diol dehydratase holoenzyme is known to undergo inactivation by
O2 in the absence of substrate (30). This inactivation is considered because of reaction of the activated Co
C bond of the enzyme-bound coenzyme with O2. Fig. 2B shows that the
O2-inactivated holoenzyme also undergoes reactivation by
the factor in the presence of AdoCbl, ATP, and Mg2+. The
extent of reactivation increased with time of incubation and reached at
least 71% at 20 min. Again, the reactivation was strictly dependent on
the factor and on ATP/Mg2+ and free AdoCbl.
In Vitro Activation of the Enzyme·CN-Cbl Complex by the
Reactivating Factor--
The capability of the reactivating factor to
activate the inactive complex of diol dehydratase with CN-Cbl was also
examined in vitro because the inactive enzyme·CN-Cbl
complex can be considered as a model of the inactivated holoenzyme.
Fig. 3A indicates that the
enzyme·CN-Cbl complex is rapidly activated by the factor in the
presence of AdoCbl, ATP, and Mg2+. This activation by the
factor also required ATP/Mg2+ (Fig. 3A) in
addition to free AdoCbl (data not shown). Approximately 76% of the
enzyme·CN-Cbl complex underwent activation by 20 min of incubation
under the conditions. As shown in Fig. 3B, the extent of
reactivation was dependent on a molar ratio of the factor to the
enzyme·CN-Cbl complex. From the double-reciprocal plot, the concentration of the factor giving half-maximal activation of 1.2 µM enzyme·CN-Cbl complex was calculated to be 3.5 µM.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Activation of diol dehydratase·CN-Cbl
complex by the reactivating factor. A, enzyme·CN-Cbl
complex was prepared by incubation of apoenzyme (12.6 units) with 11 µM CN-Cbl at 37 °C for 30 min in 0.18 ml of 0.05 M potassium phosphate buffer (pH 8). The enzyme·CN-Cbl
complex (1.5 units) was incubated at 37 °C for the indicated time
periods with ( , ) and without ( , ) 47 µg of the
reactivating factor in 50 µl of 0.02 M potassium
phosphate buffer (pH 8) containing 21 µM AdoCbl and 1.2 M 1,2-propanediol in the presence ( , ) and absence
( , ) of 24 mM ATP/24 mM MgCl2
as described in the legend to Fig. 2. Time course of 1,2-propanediol
dehydration with non-inactivated enzyme was measured as a control
( ). The amount of propionaldehyde formed was determined after
appropriate dilution. B, enzyme·CN-Cbl complex was
prepared by incubation of apoenzyme (1.5 units) with 26 µM CN-Cbl at 37 °C for 30 min in 9 µl of 0.04 M potassium phosphate buffer (pH 8). The enzyme·CN-Cbl
complex (1.5 units) was incubated at 37 °C for 10 min with the
indicated amount of the reactivating factor in 50 µl of 0.03 M potassium phosphate buffer (pH 8) containing 21 µM AdoCbl, 0.8 M 1,2-propanediol, 24 mM ATP, and 24 mM MgCl2. The amount
of propionaldehyde formed was determined after appropriate dilution.
Inset, double reciprocal plot (1/v
versus 1/[reactivating factor]).
|
|
Direct Evidence for Cobalamin Exchange--
The reactivating
factor, free AdoCbl, ATP, and Mg2+ were absolutely required
for both reactivation of the glycerol-inactivated holoenzyme and
activation of the enzyme·CN-Cbl complex. ADP was not able to replace
ATP. From the absolute requirement for free AdoCbl, it was
strongly suggested that the reactivation of the glycerol-inactivated holoenzyme and activation of the
enzyme·CN-Cbl complex occurs by exchange of the enzyme-bound,
modified coenzyme and CN-Cbl, respectively, for free intact AdoCbl. We
have previously reported that the in situ reactivation of
the glycerol-inactivated holoenzyme or the in situ
activation of inactive cobalamin-enzyme complexes takes place by
exchange of enzyme-bound cobalamins for AdoCbl (13, 18).
To examine whether the reactivating factor mediates such exchange, the
enzyme·CN-Cbl complex was subjected to incubation with and without
the reactivating factor in the presence of AdePeCbl, ATP, and
Mg2+, followed by dialysis to remove unbound cobalamins.
AdePeCbl, an inactive analog of AdoCbl containing the adenine ring in
the upper axial ligand, was used instead of AdoCbl itself because the
complex of diol dehydratase with AdoCbl (regular holoenzyme) is
catalytically active and rather susceptible to inactivation even in the
presence of substrate (31). As depicted in Fig. 4A, the spectrum of the
dialysate indicates that the enzyme-bound CN-Cbl was replaced by
AdePeCbl with the reactivating factor. This exchange never occurred
without the factor. Thus, it is evident that the reactivating factor
mediates the exchange of the enzyme-bound CN-Cbl for free AdePeCbl. In
contrast, the reverse was not the case. That is, the replacement of the
enzyme-bound AdePeCbl by free CN-Cbl did not occur at all even with the
factor in the presence of ATP and Mg2+ (Fig.
4B). Thus, it is quite likely that the reactivating factor mediates the exchange of enzyme-bound, adenine-lacking cobalamins for
free, adenine-containing cobalamins. Because the coenzyme loses the
adenine moiety by irreversible cleavage of the Co
C bond in the
inactivation of holoenzymes by glycerol or O2, it can
therefore be concluded that the reactivating factor reactivates the
inactivated holoenzymes or activates the enzyme·CN-Cbl complex by
mediating the ATP-dependent exchange of the enzyme-bound,
modified coenzyme or CN-Cbl, i.e. adenine-lacking
cobalamins, for free intact AdoCbl, i.e. an
adenine-containing cobalamin.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 4.
The reactivating factor-mediated exchange of
the enzyme-bound cobalamin for free cobalamin in the presence of ATP
and Mg2+. Enzyme·CN-Cbl (A)
and enzyme·AdePeCbl (B) complexes were formed by
incubation of apoenzyme (100 units) with 33 µM CN-Cbl and
AdePeCbl, respectively, at 37 °C for 30 min in 0.6 ml of 0.05 M potassium phosphate buffer (pH 8). To the enzyme·CN-Cbl
(A) and enzyme·AdePeCbl (B) complexes were
added 2.5 mg ( ) or none (- - -) of the reactivating factor in 0.04 M potassium phosphate buffer (pH 8) containing 20 µM AdePeCbl (A) and 20 µM CN-Cbl
(B), 20 mM ATP, and 20 mM
MgCl2 in a total volume of 1.0 ml. The mixtures were
incubated at 37 °C for 1 h and then dialyzed at 4 °C for 2 days against 1000 volumes of 0.01 M potassium phosphate
buffer (pH 8) containing 2% 1,2-propanediol with a buffer change. The
spectra of the dialysates were measured and corrected for
dilution.
|
|
Numbers of Reactivation per Diol Dehydratase and per Reactivating
Factor--
The data shown in Figs. 2 and 3 were obtained with
1,2-propanediol as substrate for measuring enzyme activity restored.
Because 1,2-propanediol does not cause suicide inactivation at a
significant rate, there remains a possibility that the reactivating
factor may mediate only a single exchange. To test this possibility, the time course of glycerol dehydration was determined with and without
the reactivating factor. As shown in Fig.
5A, this substrate brought
about complete inactivation of the enzyme within 3 min. However, when
the reactivating factor and ATP/Mg2+ were supplemented to
the reaction mixture in addition to AdoCbl, an initial rapid phase of
glycerol dehydration was followed by a slower but almost constant rate
of the dehydration. Furthermore, when the reactivating factor was added
to the completely inactivated enzyme together with ATP/Mg2+
in the presence of free AdoCbl, the inactivated enzyme underwent rapid
reactivation. The product formed increased linearly with time of
incubation, although the rate of product formation with glycerol was
much slower than that with 1,2-propanediol. As shown in Fig.
5B, the amount of the product formed by the reactivated enzyme in 4 h was dependent on a molar ratio of the factor to diol
dehydratase. On the assumption that the probability of the mechanism-based inactivation by glycerol is not affected by the presence of the factor and ATP/Mg2+, the numbers of
reactivation per diol dehydratase and per reactivating factor were
calculated to reach 6 and 2, respectively. These numbers were 5.5 and
1.5, respectively, when the reaction was terminated at 100 min of
incubation. It is therefore evident that diol dehydratase undergoes
multiple reactivation during dehydration of glycerol. However, by
taking the dimeric subunit structure of the factor into consideration,
it was suggested that the reactivating factor failed in catalyzing
multiple exchanges of tightly bound, modified coenzyme for exogenous
AdoCbl on diol dehydratase under the experimental conditions
employed.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
Reactivation of glycerol-inactivated holodiol
dehydratase by the reactivating factor during dehydration of
glycerol. A, diol dehydratase apoenzyme (1.5 units) was
incubated at 37 °C for 2, 5, and 10 min with 130 µM
AdoCbl in 40 µl of 0.03 M potassium phosphate buffer (pH
8) containing 1.5 M glycerol. At 10 min of incubation
(arrow), 47 ( , ) or 0 ( , ) µg of the
reactivating factor was added to a reaction mixture with ( , ) and
without ( , ) 24 mM ATP/24 mM
MgCl2, in a total volume of 50 µl. The mixture was
further incubated at 37 °C for the indicated time periods. In one
experiment, the reactivating factor, ATP, and MgCl2 were
added to the reaction mixture at the start of the reaction ( ). The
amount of -hydroxypropionaldehyde formed was determined after
appropriate dilution. B, diol dehydratase apoenzyme (1.5 units) was incubated with 100 µM AdoCbl at 37 °C for
4 h together with and without the indicated amount of reactivating
factor in 50 µl of 0.02 M potassium phosphate buffer (pH
8) containing 1.2 M glycerol in the presence ( ) and
absence ( ) of 24 mM ATP/24 mM
MgCl2. The amount of -hydroxypropionaldehyde formed was
determined after appropriate dilution. The numbers of reactivation per
diol dehydratase ( ) and per reactivating factor ( ) are shown in
the inset.
|
|
 |
DISCUSSION |
AdoCbl-dependent enzymatic reactions are initiated by
homolysis of the Co
C bond of the enzyme-bound coenzyme and proceed via radical mechanisms (1, 3, 4, 32-36). Such reactions are considered
to need the assistance of high reactivity of a free radical. Highly
reactive radical intermediates must sustain their reactivity at the
active sites and become extinct in the only way destined for the
reaction. Once a radical intermediate becomes extinct or stabilized by
side reactions, regeneration of AdoCbl becomes impossible, resulting in
irreversible modification of the coenzyme (3). This leads to
inactivation of enzymes because the modified coenzymes remain tightly
bound to enzymes and not exchangeable with free AdoCbl. Are such
inactivated enzymes reactivated? This would be important for cellular
economics of energy. The data presented in this paper gave at least one
answer to this question. That is, the purified complex of the DdrA and DdrB proteins reactivates glycerol-inactivated and
O2-inactivated holoenzymes. This result was as expected
because the ddrAB genes were identified as the genes of
K. oxytoca encoding a putative reactivating factor for
inactivated diol dehydratase (20). This is the first biochemical
demonstration of such reactivation by these gene products.
We have previously demonstrated with toluene-treated cells of K. oxytoca and K. pneumoniae (18) and recombinant E. coli harboring plasmid pUCDD11 (19) that the complexes of diol
dehydratase or glycerol dehydratase with CN-Cbl, aquacobalamin, and
pentylcobalamin undergo in situ activation, whereas the
complexes with adeninylbutylcobalamin and AdePeCbl do not. These facts
suggest that the affinity of the enzyme for cobalamins lacking the
adenine moiety in the upper axial ligand is selectively lowered
in situ in the presence of ATP and Mg2+,
resulting in replacement of these enzyme-bound cobalamins by free
AdoCbl. It seemed likely that the binding affinity for the modified
coenzymes in the glycerol-inactivated and O2-inactivated holoenzymes is also lowered in situ in the presence of ATP
and Mg2+ because the adenosyl group is irreversibly severed
from the cobalamin moiety during the inactivation processes (4, 14,
30). Evidence for such an exchange mechanism of reactivation was
obtained here in vitro for the first time. It was
demonstrated that the reactivating factor reactivates the inactivated
holoenzyme or activates the enzyme·CN-Cbl complex by mediating the
exchange of the enzyme-bound, adenine-lacking cobalamin for free
AdoCbl, an adenine-containing cobalamin.
Because the adenine-lacking cobalamins are also bound by diol
dehydratase so tightly, such exchange never occurs without the reactivating factor. Complex formation between the factor and diol
dehydratase was observed (data not shown). Therefore, it can be
postulated that the reactivating factor selectively lowers the affinity
of diol dehydratase for adenine-lacking cobalamins by forming a complex
with the enzyme protein and transiently affecting its higher-order
structures. The free energy required for such structural transitions
may be provided by coupling with the hydrolysis of ATP because the
reactivating factor shows low but distinct ATPase activity (data not
shown). Non-hydrolyzable ATP analogs were not effective for the
reactivation of the glycerol-inactivated holoenzyme and the activation
of the enzyme·CN-Cbl complex. Such mechanism of action of the
reactivating factor seems similar to those of molecular chaperones. In
this sense, the diol dehydratase-reactivating factor may be considered
as a new type of molecular chaperone which is involved in reactivation
of inactivated enzymes. Detailed mechanism of action of the factor is
under current investigation.
The proteins homologous to the Ddr proteins were assumed to serve as a
reactivating factor for inactivated glycerol dehydratase (20).
Therefore, such reactivating factor may be a factor of general
importance for AdoCbl-dependent enzymes.