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-fo
Trang 1for adenosylcobalamin-dependent diol and glycerol
dehydratases
Hideki Kajiura1, Koichi Mori1, Naoki Shibata2and Tetsuo Toraya1
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 B12
)-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 B12; 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: toraya@cc.okayama-u.ac.jp
(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 adeninylpentylcobalacyanocobala-min, 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-dehydratase-reactivating factor; MBTH, 3-methyl-2-benzothiazolinone hydrazone.
Trang 2b-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 O2 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 O2 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 Mg2+ (or
Mn2+) [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 dehydradehydra-tase-reacti-
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 a2b2 complex and actu-ally function as their reactivating factor – that is, they reactivate the glycerol-inactivated and O2-inactivated holoenzymes and activate the inactive enzyme–CN-Cbl complexes in vitro in the presence of AdoCbl, ATP, and Mg2+[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)2structures –
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
Trang 3more 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 holo-
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 Mg2+, 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 Mg2+ (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
Mg2+, 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 Mg2+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 l M AdoCbl and 1.2 M 1,2-propanediol in the presence of 24 m M ATP⁄ 24 m M
MgCl2, 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.
Trang 4reactivation 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 Mg2+ 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 Mg2+ 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 cobalcobal-amin 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-dehydratase–CN-Cbl (E,F), diol dehydratase–AdePedehydratase–CN-Cbl (C,D), and glycerol dehydratase–AdePedehydratase–CN-Cbl (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 l M AdePeCbl (A,B,E,F) or CN-Cbl (C,D,G,H) and 20 m M ATP ⁄ 20 m M
MgCl2, 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 m M ATP ⁄ 24 m M MgCl2, in a
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.
Trang 5exchange 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
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 m M MgCl2(B,E) or 21 m M
ATP ⁄ 21 m M MgCl2(C,F) in 35 m M 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 ⁄ 1 m M MgCl 2 (B,E) or 1 m M ATP ⁄ 1 m M 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.
Trang 6of 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
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 m M MgCl 2 in 35 m M 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 ⁄ 1 m M MgCl2(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.
Trang 7bases 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 (sapoglycer-olid 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 m M MgCl2(C,G) or 10 m M ATP ⁄ 10 m M MgCl2(D,H) in 38 m M
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 m M ) were added to 0.2 mL of the diluted mixtures without (open circles) and with (closed cir-cles) additional ATP and MgCl2(10 m M 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.
Trang 8nucleotide 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–DDR¼ 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 subsub-units 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 B12
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 37C for 10 min After the reac-tion was terminated by adding 1 mL of 0.1 m potassium citrate buffer (pH 3.6), MBTHÆHCl was added to a final concentration of 0.9 mm, and the mixture was incubated again at 37C 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)1at 37C under the standard assay conditions Protein concentrations of purified enzymes and reactivat-ing factors were determined by measurreactivat-ing 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)1Æcm)1 for diol dehydratase [35], 112 100
m)1Æcm)1for glycerol dehydratase [49], 58 140 m)1Æcm)1for DDR [35], and 86 500 m)1Æcm)1for 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
37C 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,
Trang 9followed by dialysis at 4C 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 37C 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
37C 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 MgCl2, 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 MgCl2 (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 37C for 30 min in 22.5 lL of 33 mm
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
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