Although the reactivase hydrolyzed ATP to some extent even in the absence of divalent metal ions, Mg2+, Mn2+, Co2+ and Ni2+ enhanced the ATPase activity by 3.7–4.5 fold at 3 mm.. The enz
Trang 1reactivase – evidence for multiple turnovers and subunit swapping with diol dehydratase
Koichi Mori, Yasuhiro Hosokawa, Toshiyuki Yoshinaga and Tetsuo Toraya
Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Japan
Keywords
adenosylcobalamin; coenzyme B12; diol
dehydratase; diol dehydratase-reactivating
factor; reactivase
Correspondence
T Toraya, Department of Bioscience and
Biotechnology, Graduate School of Natural
Science and Technology, Okayama
University, Tsushima-naka, Kita-ku,
Okayama, 700-8530, Japan
Fax: +81 86 251 8264
Tel: +81 86 251 8194
E-mail: toraya@cc.okayama-u.ac.jp
(Received 26 July 2010, revised
17 September 2010, accepted 1 October
2010)
doi:10.1111/j.1742-4658.2010.07898.x
Adenosylcobalamin-dependent diol dehydratase (DD) undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co–C bond The damaged cofactor remains tightly bound to the active site The DD-reactivating factor reacti-vates the inactivated holoenzyme in the presence of ATP and Mg2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme In this study, we demonstrated that this reactivating factor mediates the cobalamin exchange not stoichiometrically but catalyti-cally in the presence of ATP and Mg2+ Therefore, we concluded that the reactivating factor is a sort of enzyme It can be designated DD reactivase The reactivase showed broad specificity for nucleoside triphosphates in the activation of the enzymeÆcyanocobalamin complex This result is consistent with the lack of specific interaction with the adenine ring of ADP in the crystal structure of the reactivase The specificities of the reactivase for divalent metal ions were also not strict DD formed 1 : 1 and 1 : 2 com-plexes with the reactivase in the presence of ADP and Mg2+ Upon com-plex formation, one b subunit was released from the (ab)2 tetramer of the reactivase This result, together with the similarity in amino acid sequences and folds between the DD b subunit and the reactivase b subunit, suggests that subunit displacement or swapping takes place upon formation of the enzymeÆreactivase complex This would result in the dissociation of the damaged cofactor from the inactivated holoenzyme, as suggested by the crystal structures of the reactivase and DD
Structured digital abstract
l MINT-7997177: Reactivase alpha (uniprotkb:O68195), Reactivase beta (uniprotkb:O68196), Diol Dehydratase gamma (uniprotkb:Q59472), Diol Dehydratase beta (uniprotkb:Q59471) and Diol Dehydratase alpha (uniprotkb:Q59470) physically interact (MI:0915) by comigration in non denaturing gel electrophoresis (MI:0404)
l MINT-7997157: Diol Dehydratase alpha (uniprotkb:Q59470), Diol Dehydratase beta (uni-protkb:Q59471), Diol Dehydratase gamma (uniprotkb:Q59472), Reactivase beta (uniprotkb: O68196) and Reactivase alpha (uniprotkb:O68195) physically interact (MI:0915) by molecular sieving (MI:0071)
Abbreviations
AdePeCbl, adeninylpentylcobalamin; AdoCbl, adenosylcobalamin or coenzyme B12; CN-Cbl, cyanocobalamin; DD, diol dehydratase.
Trang 2Adenosylcobalamin (AdoCbl)-dependent enzymes
cata-lyze chemically difficult reactions by the use of highly
reactive radicals The homolytic cleavage of the Co–C
bond of the coenzyme forms a Co(II) species and an
adenosyl radical, which triggers the reactions [1]
Although enzymes generally deal with highly reactive
intermediates by ‘negative catalysis’ [2], cobalamin
enzymes tend to undergo mechanism-based
inactiva-tion because of the involvement of highly reactive
radi-cal intermediates during catalysis [3] Diol dehydratase
(DD) (EC 4.2.1.28) catalyzes the AdoCbl-dependent
conversion of 1,2-propanediol, glycerol and
1,2-ethane-diol to the corresponding aldehydes [4,5] Its
physio-logical substrates are 1,2-diols, such as 1,2-propanediol
[6,7], but it functionally substitutes for glycerol
dehy-dratase (EC 4.2.1.30), an isofunctional enzyme of DD,
in the anaerobic dissimilation of glycerol by
Klebsiel-la oxytoca and some other bacteria that lack glycerol
dehydratase [8,9] Despite their roles, both enzymes
undergo mechanism-based inactivation by glycerol
[5,10–12], accompanying the irreversible cleavage of
the Co–C bond of the enzyme-bound coenzyme The
damaged cofactor thus formed remains tightly bound
to the apoenzyme and is not displaced by intact
Ado-Cbl, resulting in the irreversible inactivation of the
enzyme
This apparent inconsistency was resolved by our
finding of the rapid reactivation of glycerol-inactivated
enzymes in permeabilized Klebsiella pneumoniae and
K oxytocacells (in situ) [13,14] Specific protein factors
that are responsible for the reactivation of the
inactivated holoenzymes of DD [15–17] and glycerol
dehydratase [18–20] were found, and designated
DD-reactivating factor and glycerol
dehydratase-reactivating factor, respectively They reactivated the
O2-inactivated [16,19,20] and
3-butene-1,2-diol-inacti-vated [21,22] holoenzymes as well We demonstrated
that these factors reactivate the inactivated
holoen-zymes by a molecular chaperone-like mechanism
(Fig 1) [16,17,19,23] Salient features are as follows
The reactivating factor binds ATP and hydrolyzes it to
ADP by its own weak ATPase activity The resulting
ADP-bound form of the reactivating factor has a high
affinity for the enzyme, and interacts with the
inacti-vated holoenzyme to form a tight
apoenzymeÆreactivat-ing factor complex, with the concomitant release of the
damaged cofactor The reactivating factor reverts to a
low-affinity form through the replacement of bound
ADP by free ATP, resulting in the dissociation of the
apoenzymeÆreactivating factor complex into apoenzyme
and the reactivating factor Active holoenzyme is then
reconstituted from apoenzyme and free AdoCbl DD does not form a complex with the reactivating factor while it exists as an active holoenzyme The glycerol dehydratase-reactivating factor reactivates the inacti-vated hologlycerol dehydratase in a similar manner Both dehydratase-reactivating factors exist as a2b2 heterotetramers [a, DdrA or GdrA (DhaF); b, DdrB or GdrB (DhaG)] [16,19,20] Liao et al reported the crystal structure of the nucleotide-free form of glycerol dehydratase-reactivating factor [24] Independently, we solved the crystal structures of the DD-reactivating factor in both the ADP-bound and nucleotide-free forms [25] The structures of both reactivating factors are similar Their a subunits have a structural feature common to the ATPase domains of actin superfamily proteins, including Hsp70 molecular chaperones Interestingly, their b subunits have similar folds to the
b subunits of diol and glycerol dehydratases Such structural characteristics provide important clues to help solve the mechanisms of action of these reactivat-ing factors – that is, subunit swappreactivat-ing might occur However, no biochemical evidence for this has been obtained so far A similar reactivating factor for ethanolamine ammonia lyase has been reported [26]
It has also been reported that a protein named E2 activates lysine-5,6-aminomutase in an ATP-dependent manner, although its exact function is not yet known [27]
In this study, we examined whether and how the complexes between DD and its reactivating factor are formed Specificities of the reactivating factor for nucleotides and divalent cations were also investi-gated In addition, it was determined whether the reactivating factor-mediated cobalamin release is catalytic
Fig 1 Mechanism of the reactivation of inactivated holoenzymes
by reactivating factors E, DD or glycerol dehydratase; RF, DD-reac-tivating factor or glycerol dehydratase-reacDD-reac-tivating factor; X-Cbl, a damaged cofactor; AdoH, 5¢-deoxyadenosine.
Trang 3Evidence for multiple turnovers of
DD-reactivating factor in cobalamin exchange
In a previous article, we reported the number of the
reactivating factor-mediated reactivations of DD
during the dehydration of glycerol [16] The number of
reactivations per molecule of DD was calculated to be
approximately six under conditions where the
reacti-vating factor was added to a 10-fold molar excess
rela-tive to the enzyme This indicates that the enzyme
undergoes multiple inactivation–reactivation cycles On
the other hand, the maximum number of reactivations
per molecule of the reactivating factor was observed to
be approximately two, at a molar ratio of the
reacti-vating factor to the enzyme of 0.5 As the reactireacti-vating
factor exists as a dimer of ab heterodimers, i.e (ab)2,
it remained unclear whether the reactivating
factor-mediated reactivation of inactivated holoenzymes is
catalytic or stoichiometric It is experimentally not
possible to demonstrate multiple turnovers for the
reactivating factor in this reactivation assay, probably
because of the inhibition of the holoenzyme by
accu-mulated 3-hydroxypropionaldehyde
To avoid this difficulty, we examined whether the
reactivating factor can mediate multiple turnovers of
the replacement of tightly bound cyanocobalamin
(CN-Cbl) (an inactive coenzyme analog lacking the
ade-nine ring in the upper axial ligand; a model of damaged
cofactors) for free adeninylpentylcobalamin (AdePeCbl)
(an inactive coenzyme analog containing the adenine
ring in the upper axial ligand; a model of intact
coen-zyme, AdoCbl) in the presence of ATP and Mg2+at a
molar ratio of the reactivating factor to the enzyme of
0.1 (Fig 2) The spectrum of the enzyme obtained after
the removal of unbound cobalamins indicated that the
enzyme-bound CN-Cbl was replaced by AdePeCbl in a
manner dependent on both the reactivating factor and
ATP⁄ Mg2+ (Fig 2A,B) Figure 2C shows the time
course of the exchange of enzyme-bound CN-Cbl for
AdePeCbl in the presence of the reactivating factor and
ATP⁄ Mg2+ About 70% of the enzyme-bound CN-Cbl
was replaced with AdePeCbl within 1 h, and the
replacement was almost complete within 4 h The total
amount of enzyme-bound cobalamin remained almost
constant (1.9–2.1 molÆmol)1) during incubation It is
thus evident that the reactivating factor mediates the
exchange of enzyme-bound CN-Cbl for free AdePeCbl
for a 10-fold molar excess of the enzyme under the
conditions employed This strongly suggests that
the reactivation of the inactivated holoenzymes by the
factor is not stoichiometric but catalytic Hence, the
A
B
C
Fig 2 Evidence for the catalytic turnover of the DD-reactivating factor (A) The reactivating factor-mediated replacement of enzyme-bound CN-Cbl with free AdePeCbl in the presence of ATP and
Mg 2+ was analyzed by the spectral change of enzyme-bound cobal-amin A 10-fold excess of the enzymeÆCN-Cbl complex over the reactivating factor was used Experimental details are described in the text After removal of unbound cobalamin at 0 min (thick solid line), 30 min (thin solid line), 60 min (thin long-dashed line),
120 min (thin short-dashed line), 240 min (thin dotted line) and
360 min (thick dotted line) of incubation, the spectrum of enzyme-bound cobalamin was measured Inset: spectra of enzyme-enzyme-bound CN-Cbl (solid line) and AdePeCbl (dotted line) (B) Experimental con-ditions were the same as in (A), except that spectra were taken after 360 min of incubation in the absence of ATP and Mg 2+ (thick solid line) or without the reactivating factor in the presence of ATP and Mg2+(thick dotted line) (C) Time course of the reactivating fac-tor-mediated exchange of enzyme-bound CN-Cbl for AdePeCbl The extent of exchange was determined from the change in absor-bance at 364 nm The total amount of enzyme-bound cobalamin was determined spectrophotometrically after conversion to the dicyano form Inset: a semilogarithmic plot.
Trang 4DD-reactivating factor can be designated DD reactivase
as well The cobalamin exchange occurs through the
intermediary formation of apoenzyme [17,19] As
Ade-PeCbl binding to apoenzyme takes place much faster
than CN-Cbl release, the rate of CN-Cbl replacement
can be considered to be the rate of CN-Cbl release The
inset of Fig 2C indicates that the rate of CN-Cbl
replacement (release) follows pseudo-first-order
kinet-ics, and the rate constant of the reactivase in cobalamin
release (kcat,cbl-release) for CN-Cbl was calculated to be
0.27 min)1at 37C from the initial rate
The time course of the reactivation of
glycerol-inacti-vated holoenzyme by the reactivase in the presence of
ATP and Mg2+at a molar ratio of the reactivase to the
enzyme of 0.1 is shown inFig 3 From the initial rate,
the rate constant of the reactivase in the reactivation
(kcat,react) was calculated to be 0.071 ± 0.008 min)1 at
37C Considering that the enzyme contains two
cobal-amin-binding sites in the (abc)2 dimer, it can be
assumed that the reactivase mediates the exchange of
enzyme-bound damaged cofactor for intact AdoCbl
with a rate constant (kcat,cbl-release) of 0.14 min)1
Kinetic parameters of the reactivase for ATP in
DD (re)activation and ATP hydrolysis
Kinetic constants for ATP in the reactivation of
glyc-erol-inactivated holoenzyme and the activation of the
enzymeÆCN-Cbl complex by the reactivase were mea-sured (Table 1) Kmvalues for ATP in the reactivation and the activation were essentially the same: 6.9 ± 0.4 mm and 6.8 ± 1.6 mm, respectively This is reasonable, because these two events are different aspects of the same phenomenon [14,16] Kmvalues for the ATPase activity were also measured in the presence and absence of equimolar apoenzyme (Table 1) The Km for ATP in the ATPase activity in the absence of enzyme was 61 ± 14 lm, i.e two orders of magnitude smaller than that in the DD (re)activation Moreover, the Km for ATP in the ATPase activity was essentially not affected by the presence of enzyme (67 ± 17 lm) The kcat in the ATPase activity was estimated to be 1.4 ± 0.1 min)1 in the absence of apoenzyme The ATPase activity was slightly inhibited by the presence of apoenzyme (kcat= 1.2 ± 0.1 min)1) These values are
in good agreement with those previously reported [17]
Nucleotide and divalent cation specificities of the reactivase
The specificities of the reactivase for nucleotides in the activation of the enzymeÆCN-Cbl complex were studied
in the presence of AdoCbl and Mg2+ (Table 2) Although ATP was most effective (40% as compared with the apoenzyme control), CTP, UTP and GTP showed comparable efficiencies (82%, 75% and 55% relative to ATP, respectively) Moreover, 2¢-dATP and 3¢-dATP were 55% and 63% as effective as ATP The efficiencies of various divalent metal ions for the activation of the enzymeÆCN-Cbl complex by the reactivase were also examined at 3 mm (Table 2) The
Fig 3 Time course of the reactivation of the glycerol-inactivated
holoenzyme by DD reactivase The glycerol-inactivated holoenzyme
formed as described in the text was subjected to ultrafiltration on a
Microcon YM-10 microconcentrator (Millipore) To a concentrated
protein fraction containing 1.2 nmol of glycerol-inactivated
holoen-zyme, we added 2.3 M 1,2-propanediol, 38 l M AdoCbl, 19 m M ATP
and 19 m M MgCl 2 in 0.02 M potassium phosphate buffer (pH 8.0)
without or with 0.12 nmol of reactivase to a total volume of
160 lL After incubation at 37 C for the indicated time periods,
20 lL aliquots were withdrawn, and the amount of DD reactivated
was measured by the 3-methyl-2-benzothiazolinone hydrazone
method [33] after appropriate dilution.
Table 1 Kinetic parameters of the reactivase for ATP.
Kmfor ATP (m M )
V max (lmol propionaldehyde formed in 10 min) kcat(min)1) Reactivation a 6.9 ± 0.4 2.2 ± 0.6
Activation a 6.8 ± 1.6 17 ± 2
a The glycerol-inactivated holoenzyme (31 pmol) or the enzymeÆCN-Cbl complex (53 pmol) was incubated at 37 C for 10 min with 0.21 nmol of reactivase in 70 lL of 0.02 M potassium phosphate buf-fer (pH 8.0) containing 0.6 M 1,2-propanediol, 0.01 M KCl, and 21 l M
AdoCbl, with 0–40 m M each of ATP and MgCl2 The reaction was terminated by addition of 70 lL of 0.1 M potassium citrate buffer (pH 3.6) The amount of propionaldehyde formed was determined as described in the text after appropriate dilution b The reactivase (0.22 nmol) was incubated at 37 C for 5 min with or without apoen-zyme (5.0 units, 0.20 nmol) in 50 lL of 0.01 M potassium phosphate buffer (pH 8.0) containing 0.3–10 m M each of [ 32 P]ATP[cP] and MgCl2 ATPase activity was measured as described in the text.
Trang 5divalent cations tested did not inhibit the DD activity at
3 mm in the standard assay conditions (data not shown)
Mn2+ was most effective for the activation (81% as
compared with the apoenzyme control; 185% relative to
Mg2+) Co2+ and Ni2+ were also effective (97% and
48%, respectively, relative to Mg2+), whereas Ca2+,
Cr2+ (Cr3+) and Cu2+ were not (< 2% relative to
Mg2+) Mg2+ was used routinely in the (re)activation
assay, because it is a physiological divalent metal ion
The specificity of the reactivase for divalent metal ions
in ATP hydrolysis was also measured (Table 3)
Although the reactivase hydrolyzed ATP to some extent
even in the absence of divalent metal ions, Mg2+,
Mn2+, Co2+ and Ni2+ enhanced the ATPase activity
by 3.7–4.5 fold at 3 mm On the other hand, Ca2+, Cr2+
(Cr3+) and Cu2+had little or no effect on the ATPase
activity as compared with the control without divalent
metal ions Irrespective of the presence of divalent
cations, ATP was hydrolyzed to ADP + Piby the
reac-tivase (data not shown)
Analysis of complex formation between DD and
its reactivase by gel filtration
Apoenzyme was incubated with the reactivase in the
presence of ADP or ATP and Mg2+, and then subjected
to gel filtration on a Superose 6 column that had been preliminarily equilibrated with nucleotide⁄ Mg2+ -containing buffer (Fig 4) In the presence of ATP, the enzyme and the reactivase eluted separately at their respective retention times In contrast, a peak of the free reactivase decreased and a new peak appeared in the presence of ADP The latter peak was eluted with a
Table 2 Nucleotide and divalent cation specificities of the reactivase for the activation of the enzymeÆCN-Cbl complex The enzymeÆCN-Cbl complex (DDÆCN-Cbl) (61 pmol) was incubated at 37 C for 10 min with and without 0.30 nmol of reactivase in 50 lL of 0.02 M potassium phosphate buffer (pH 8.0) containing 21 l M AdoCbl and 1.2 M 1,2-propanediol in the presence and absence of 3 m M ATP (or an indicated nucleotide) and 3 m M MgCl2(or a chloride salt of the indicated divalent metal ions) The reaction was terminated by addition of 50 lL of 0.1 M potassium citrate buffer (pH 3.6) The amount of propionaldehyde formed was determined as described in the text after approriate dilution.
Propionaldehyde formed
Relative activity (%)
a Cr 2+ added might be oxidized to Cr 3+ by air in the reaction mixture.
Table 3 Divalent cation specificity of the reactivase for the ATPase activity The reactivase (0.22 nmol) was incubated at 37 C for
30 min with 3 m M [ 32 P]ATP[cP] in 50 lL of 0.01 M potassium phos-phate buffer (pH 8.0) in the presence and absence of the indicated divalent metal chloride (3 m M ) The ATPase activity was measured
as described in the text.
Metal ion
ATPase activity (min)1)
Relative activity (%)
a Cr 2+ added might be oxidized to Cr 3+ by air in the reaction mixture.
Trang 6retention time that was slightly shorter than that of the
free enzyme and much shorter than that of the
reactivas-e When this peak was subjected to SDS⁄ PAGE, the
peak comprised all of the subunits from the enzyme
(a, b, and c) and the reactivase (a and b) (data not
shown) It was thus evident that this peak contained the
enzymeÆreactivase complex(es) in addition to a small
amount of the free enzyme In fact, when this peak was
analyzed by nondenaturing PAGE, two bands of the
enzymeÆreactivase complexes described below were
detected (data not shown) A similar peak containing
the enzymeÆreactivase complexes was observed in the
absence of nucleotide as well, although the content of
complexes was rather lower than in the presence of
ADP
Characterization of the enzymeÆreactivase
complexes by nondenaturing PAGE
When the enzymeÆCN-Cbl complex was incubated with
the reactivase in the presence of ADP and Mg2+ and
followed by nondenaturing PAGE, the bands of the
enzyme and the reactivase were markedly reduced
in density, and two bands of the enzymeÆreactivase
complexes (upper and lower) appeared above them
(Fig 5A, lane c) When apoenzyme was used instead of
the enzymeÆCN-Cbl complex, both bands of the
enzymeÆreactivase complexes were observed in the
presence of ADP (Fig 5A, lane e) The abundance of
the upper band relative to the lower band of the enzymeÆreactivase complex formed from apoenzyme was significantly larger than that formed from the
Fig 4 Analysis of interaction between DD and its reactivase by
gel filtration column chromatography Experimental details are
described in the text 1, apoenzyme + reactivase (+ADP); 2,
apoen-zyme (+ADP); 3, reactivase (+ADP); 4, apoenapoen-zyme + reactivase
(+ATP) Positions of the free apoenzyme (DD), reactivase (DD-R)
and the enzymeÆreactivase complex(es) (DDÆDD-R) are indicated on
the tops of chromatograms.
A
B
C
66 K–
66 K–
45 K–
36 K–
29 K–
20 K–
14 K–
Fig 5 Analysis of the enzymeÆreactivase complex by PAGE Exper-imental details are described in the text (A) Nondenaturing PAGE
in the presence of ADP and Mg 2 +
Lanes: (a) apoenzyme; (b) reacti-vase; (c) enzymeÆCN-Cbl complex + reactivase (+ADP); (d) enzy-meÆCN-Cbl complex (+ADP); (e) apoenzyme + reactivase (+ADP); (f) apoenzyme (+ADP); (g) reactivase (+ADP) +ADP indicates that samples were incubated with ADP ⁄ MgCl 2 (B, C) Bands i–vi in (A) were excised and subjected to SDS ⁄ PAGE on 6% (B) and 14% (C) gels Lanes i–vi correspond to bands i–vi in (A) Lane D: purified enzyme Lane R: purified reactivase Positions of DD and its subun-its (aD, bDand cD) and the reactivase (DD-R) and its subunits (aR and b R ) are indicated on the right of the gels In (C), to improve visi-bility of the bands of small subunits, especially bR, three excised pieces of the same bands from nondenaturing PAGE were sub-jected together to SDS ⁄ PAGE This resulted in saturation of the bands of large subunits, i.e a D and a R Densitometric analysis was carried out with other gels in which such saturation did not occur (not shown).
Trang 7enzymeÆCN-Cbl complex These results are consistent
with previous data [17] Three bands were clearly seen in
the lanes containing the free reactivase, i.e two adjacent
thick bands and one thin band on the front line,
irre-spective of the presence of enzyme (Fig 5A, lanes b, c,
e, and g) To determine their subunit compositions,
bands i–vi were excised and subjected to SDS⁄ PAGE
on 6% and 14% gels (Fig 5B,C), followed by
densito-metric analyses Both band i and band vi of the
enzymeÆreactivase complexes comprised all of the
subunits from the enzyme (a, b, and c) and the
reacti-vase (a and b) (Fig 5B,C, lanes i and vi) Subunit
compositions of the bands were the same when either
the enzymeÆCN-Cbl complex or apoenzyme was used
(data not shown) If the a, b and c subunits of the
enzyme are abbreviated as aD, bD, and cD, respectively,
and the a and b subunits of the reactivase are
abbrevi-ated as aRand bR, respectively, molar ratios of aD, bD,
cD, aRand bRin bands i and vi were determined to be
about 2 : 2 : 2 : 2 : 1 and 1 : 1 : 1 : 2 : 1, respectively,
by densitometric analysis Therefore, it was
demon-strated that bands i and vi are (aDbDcD)2Æ(aRÆaRbR) and
(aDbDcD)2Æ(aRÆaRbR)2 complexes, respectively We
named the former the enzymeÆreactivase (1 : 1) complex
and the latter the enzymeÆreactivase (1 : 2) complex
When bands ii and iii of Fig 5A, i.e two thick bands of the reactivase, were subjected to SDS⁄ PAGE, they con-tained both aRand bR, although the ratios of subunits were different (Fig 5C, lanes ii and iii) Densitometric analysis indicated that molar ratios of aR to bR for bands ii and iii were approximately 2 : 1 and 1 : 1, respectively These results indicated that the upper and lower bands of the reactivase correspond to the aRÆaRbR and (aRbR)2complexes, respectively The thin band on the front line of nondenaturing PAGE (Fig 5A, band iv) contained only bR (Fig 5C, lane iv) It possibly represents a monomer of bR, as there is no direct interaction between two adjacent bR subunits in the crystal structure of the (aRbR)2 tetramer of the reacti-vase [25] In the absence of ADP⁄ Mg2+ or in the presence of ADP but in the absence of Mg2+, the enzymeÆreactivase complex was formed in small amounts from apoenzyme and the reactivase and not at all from the enzymeÆCN-Cbl complex and the reactivase (data not shown)
Affinity of the reactivase for DD
Figure 6 shows the dependence of enzymeÆreactivase complex formation on reactivase concentration The
A
B
Fig 6 Dependence of complex formation
on reactivase concentration at a fixed
enzyme concentration (A) Apoenzyme +
reactivase (left, none; right, +ADP).
(B) EnzymeÆCN-Cbl complex + reactivase
(left, none; right, +ADP) Experimental
condi-tions were similar to those for nondenaturing
PAGE in Fig 5, except that the reactivase
and enzyme concentrations were varied and
fixed (1 l M ), respectively The number on the
top of each lane indicates the reactivase
concentration (l M ) Lane R: reactivase 2 l M
Positions of the enzyme, reactivase and the
enzymeÆreactivase complexes are indicated
on the right of the gels: (i) enzymeÆreactivase
(1 : 2) complex; (ii) enzymeÆreactivase (1 : 1)
complex; (iii) enzyme; (iv) reactivase
(a R Æa R b R ); (v) reactivase [(a R b R ) 2 ]; (vi) small
subunit of the reactivase (bR).
Trang 8enzymeÆreactivase (1 : 1) complex (band ii) was formed
even at the lowest concentration of reactivase tested
(0.25 lm) in the presence of ADP and Mg2+ In the
absence of ADP and Mg2+, it was observable at
‡ 0.5 lm reactivase Similarly, the enzymeÆreactivase
(1 : 2) complex (band i) appeared clearly at 1 lm
reacti-vase in the presence of ADP and Mg2+, whereas it was
observed at ‡ 2 lm reactivase in the absence of ADP
and Mg2+ Moreover, although the enzymeÆreactivase
(1 : 2) species was the only enzymeÆreactivase complex
observed at‡ 4 lm reactivase in the presence of ADP
and Mg2+, some enzymeÆreactivase (1 : 1) complex
remained even at the highest concentration of reactivase
tested (20 lm) in the absence of ADP and Mg2+ The
apparent KD values of the reactivase for formation of
the enzymeÆreactivase complex were 0.4 lm and 3 lm in
the presence and absence of ADP and Mg2+,
respec-tively When similar experiments were carried out with
the enzymeÆCN-Cbl complex in place of apoenzyme,
essentially no complex formation was observed, even at
20 lm reactivase, in the absence of ADP and Mg2+ In
contrast, in the presence of ADP and Mg2+, the
reacti-vase formed complexes with DD, accompanying the
release of tightly bound CN-Cbl from the enzyme [17]
However, the enzymeÆreactivase (1 : 1) complex
remained at 20 lm reactivase The apparent KD of the
reactivase was 0.7 lm
Discussion
In the present study, we demonstrated the multiple
turnovers of the DD-reactivating factor in the in vitro
activation of the inactive enzymeÆCN-Cbl complex,
and thus redesignated the reactivating factor [16,17]
DD reactivase This is reasonable in vivo from the
viewpoint of the cellular economy of energy If the
reactivating factor could not mediate the multiple
exchanges in the reactivation of inactivated
holoen-zymes, its presence would not be advantageous to the
bacterial cells We have previously demonstrated that
the hydrolysis of ATP by the reactivating factor is
catalytic [17] Moreover, the reactivation observed in
permeabilized cells of K oxytoca seems to be catalytic
[13,14], although molar ratios of the reactivating factor
to the enzyme remain obscure We previously failed to
demonstrate the multiple turnovers of the reactivase in
the reactivation of inactivated holoenzymes One
possi-ble reason for this difficulty might be the accelerated
rate of inactivation of the enzyme with
b-hydroxypro-pionaldehyde accumulating to an extremely high
concentration (20–180 mm), as the reactivation was
monitored by product formation from reactivated
holoenzyme at high concentrations of the enzyme and
the reactivase It would be easier to demonstrate the multiple turnovers of DD reactivase and glycerol dehydratase reactivase in the in situ reactivation, because the reactivation takes place in toluene-treated cells, where local concentrations of the enzyme and the reactivase are high enough for reactivation, and an inhibitory product, b-hydropropionaldehyde, diffuses away
From the initial rate of exchange of enzyme-bound CN-Cbl for AdePeCbl, the rate constant of the reacti-vase in cobalamin release (kcat,cbl-release) for CN-Cbl was calculated to be 0.27 min)1 at 37C From the initial rate of reactivation of the glycerol-inactivated holoenzyme, the rate constant of the reactivase in the reactivation (kcat,react) was calculated to be 0.071 ± 0.008 min)1 at 37C Considering that the enzyme contains two cobalamin-binding sites in the (abc)2 dimer, it can be assumed that the reactivase mediates the exchange of enzyme-bound damaged cofactor for intact AdoCbl with a rate constant (kcat,cbl-release) of 0.14 min)1 This value is about half of the above-mentioned kcat,cbl-release for CN-Cbl release This difference might be attributable to the difference
in release rate between CN-Cbl and the damaged cofactor Another possible explanation is that the enzyme activity of the reconstituted abcÆAdoCbl complex in one trimer might be affected by the neigh-boring trimer of the same enzyme molecule, i.e by the presence of the damaged cofactor or AdoCbl and their absence The kcat of the reactivase in ATP hydrolysis
in the presence of enzyme (1.2 min)1) was slightly smaller than that in its absence (1.4 min)1) It was in the same range as the rate constant of the enzyme in the suicide inactivation with glycerol (1.3 min)1) [17], but about five-fold and 10-fold larger than the rate constants for the release of CN-Cbl (0.27 min)1) and the damaged cofactor (0.14 min)1), respectively Therefore, ATP hydrolysis and cobalamin release or reactivation might be not very tightly coupled The reactivation of the inactivated holoenzymes by the reactivase seems to be physiologically relevant, because
kcat,cbl-release is much larger than the rate constant for bacterial growth on glycerol
The reactivase exhibited broad specificities for nucle-otides and divalent metal cations, both of which are absolutely required for the in vitro activation of the enzymeÆCN-Cbl complex We have previously reported similar specificities in the in situ reactivation of the glycerol-inactivated hologlycerol dehydratase with
K pneumoniae cells [13] It was established that the reactivase-mediated reactivation of the inactivated holoenzymes with ATP and Mg2+ takes place in two steps: (a) ADP-dependent cobalamin release with
Trang 9concomitant formation of the apoenzymeÆreactivase
complex; and (b) ATP-dependent dissociation of the
complex to apoenzyme and the reactivase [17] ATP
plays dual roles, i.e as a precursor of ADP in the first
step, and an effector to change the reactivase to a form
with low affinity for the enzyme The nucleotides used
in this study (ATP, GTP, CTP, UTP, 2¢-dATP, and
3¢-dATP) were effective in overall activation, although
the efficiencies were somewhat different This suggests
that these nucleotides are also effective in both steps
We determined the crystal structure of the reactivase
in the ADP-bound and nucleotide-free forms [25]
ADP is bound to the ATPase domain, a core domain
of the a subunit This domain shares common
structural features with the ATPase domain of actin
superfamily proteins, including Hsp70 molecular
chap-erones The reactivase binds ADP without specific
interactions with the adenine ring through hydrogen
bonding or base stacking The broad specificity of the
reactivase for the base moiety is thus consistent with
its crystal structure The O2¢ atom of ADP is hydrogen
bonded to the –COO)group of Glua459 and the e-NH2
group of Lysa462 These hydrogen bonds exist in the
interaction between Hsc70 and ADP as well The
resi-due corresponding to Glua459 of the DD reactivase is
Alaa461 in glycerol dehydratase reactivase
Further-more, 2¢-dATP retained half of the efficacy of ATP in
the activation of the enzymeÆCN-Cbl complex It was
therefore concluded that these hydrogen bonds are not
essential for (re)activation Similarly, 3¢-dATP retained
half of the efficacy of ATP in the activation In the
crys-tal structure of the reactivase, no amino acids were
found to be hydrogen bonded to O3¢ of ADP Thus, no
requirement for the 3¢-OH group seems to be reasonable
from its crystal structure
The reactivase has two distinct divalent metal
ion-binding sites in the ab heterodimeric unit [25] One of
them is present in the interface between the a and
b subunits This metal ion is coordinated by four
amino acids (Aspa166, Aspa183, Thra105, and
Glub31), all of which are completely conserved in both
reactivases for diol and glycerol dehydratases These
coordinations are maintained in the reactivase,
irre-spective of the ADP binding The crystal structure of
the DD reactivase suggested that this metal ion is
Mg2+ in the ADP-bound form, whereas it is Ca2+ in
the nucleotide-free form It might be possible that
Mg2+ occupies this site in vivo and is replaced by
Ca2+ in the purification of the reactivase by
hydroxy-apatite column chromatography [25] Liao et al also
reported this metal ion to be Ca2+ in the
nucleotide-free form of glycerol dehydratase reactivase that is
crystallized in the presence of Ca2+[24] In the case of
the ADP-bound form, Ca2+ in this site may be replaced by Mg2+ upon incubation of the reactivase with ADP and Mg2+ The other divalent metal ion is
Mg2+, which interacts with the b-phosphate group of ADP in the nucleotide-binding site of the a subunit This Mg2+was not found in the nucleotide-free form
Mg2+, Mn2+, Co2+ and Ni2+ enhanced the ATPase activity of the reactivase, although the reactivase can hydrolyze ATP to ADP even without divalent metal ions These metal ions were effective in the activation
of the enzymeÆCN-Cbl complex by the reactivase in the presence of ATP, although relative efficiencies were not always correlated On the other hand, the reacti-vase was unable to activate the enzymeÆCN-Cbl com-plex even in the presence of ATP with Ca2+, Cr2+or
Cu2+ or without divalent metal ions These metal ions had little or no enhancing effect on the ATPase activ-ity of the reactivase Thus, the reactivase-mediated activation of the enzymeÆCN-Cbl complex absolutely requires the hydrolysis of ATP in the presence of diva-lent metal ions The reactivase does not form the enzymeÆreactivase complexes from the enzymeÆCN-Cbl complex in the presence of ADP without divalent cations These results suggest that the binding of ADP alone to the ATPase domain of the reactivase a sub-unit is not sufficient to cause a conformational change
of the enzyme, resulting in the release of adenine-lack-ing cobalamins, such as CN-Cbl and damaged cofac-tor The fact that the relative efficiencies of metal ions for the reactivation are not always correlated with the ATPase activity of the reactivase might be attributable
to the different characteristics of binding of these diva-lent ions to the other metal ion-binding site in the interface between the a and b subunits, although the binding specificity of this site for metal ions remains unclear
We have previously demonstrated the formation of the enzymeÆreactivase complex from apoenzyme and the reactivase in the presence of ADP⁄ Mg2+or in the absence of nucleotide, although the exact subunit com-positions of the resulting complexes remained unclear Our present study indicated that two kinds of com-plexes with different subunit compositions were formed These complexes contain the enzyme and the reactivase in 1 : 1 and 1 : 2 molar ratios, and release
of the reactivase b subunit was observed upon com-plex formation (Fig 7A) These results constitute clear evidence for the displacement of the reactivase b sub-unit by the enzyme b subsub-unit (subsub-unit swapping) upon formation of the complex between the enzyme and the reactivase At present, it is not clear which complex is involved or whether both complexes are involved in the reactivation The dissociation of (aRbR)2 into
Trang 10aRÆaRbR and bR in the presence of ADP and Mg2+
was observed even without the enzyme The crystal
structure of the reactivase also suggested that the
interactions between the reactivase a and b subunits
are weakened at least partially by the ADP binding
[25] The space that is opened by the dissociation of
the reactivase b subunit would most likely be occupied
by the enzyme b subunit, as these subunits have
simi-lar folds [25,28] The docking model of the aDbDcDÆaR
complex indicates that marked steric repulsion is
induced between the enzyme a subunit and the
reacti-vase a subunit in the complex [25] The amino acid
side chains that come closer than the van der Waals
contact in the modeled structure would push each
other aside and result in tilting of the enzyme a
sub-unit with respect to the enzyme b subsub-unit Thus, it
would lead to the release of the damaged cofactor, an
adenine-lacking cobalamin, from the enzyme, because
cobalamin is bound between the a and b subunits of
the enzyme The crystal structure of DD revealed that,
like ethanolamine ammonia lyase [29], the enzyme has
a cavity 5 A˚ in height and 15 A˚ in width between
the a and b subunits (Fig 7B) The tilting of the
a subunit with respect to the b subunit upon subunit
swapping is estimated to be 6 A˚, based on the
mod-eled complex, forming a cavity 11 A˚ in height The
size of this cavity is comparable with that of
adenine-lacking cobalamins, and thus allows the damaged
cofactor to pass through it Intact cofactor, an ade-nine-containing cobalamin, is not released from the enzyme by the reactivase One reason might be its larger size, and the other possible reason might be that the additional interaction between its adenine moiety and the enzymes’s adenine-binding pocket stabilizes the interaction between the enzyme a and b subunits
In contrast, even in the absence of ADP, the reactivase forms the enzymeÆreactivase complex with apoenzyme However, it does not release the damaged cofactor from the inactivated holoenzymes under this condi-tion This may be because the steric repulsion is less
or is canceled by the conformational flexibility in the absence of ADP⁄ Mg2+ In order to prove or disprove these predictions, we have to await the structural analysis of a real enzymeÆreactivase complex
Experimental procedures
Materials Crystalline AdoCbl was a gift from Eisai (Tokyo, Japan) CN-Cbl was obtained from Glaxo Research Laboratories (Greenford, UK) AdePeCbl was synthesized according to published procedures [30] [32P]ATP[cP] was obtained from PerkinElmer (Waltham, MA, USA) 2¢-DeoxyATP and 3¢-deoxyATP were obtained from Sigma-Aldrich (St Louis,
MO, USA) All other chemicals were commercial products
of the highest grade available and were used without further purification K oxytoca recombinant DD and its reactivase were purified to homogeneity from overexpress-ing Escherichia coli JM109 harboroverexpress-ing expression plasmid pUSI2E(DD) [31] and E coli JM109 or B834 harboring expression plasmid pUSI2ENd(6⁄ 5b) [16,32], respectively,
as reported previously
Enzyme and protein assays The amount of aldehydic products formed by DD was determined by the 3-methyl-2-benzothiazolinone hydrazone method [33] One unit of the enzyme is defined as the amount of enzyme activity that catalyzes the formation of
1 lmol of propionaldehyde per minute at 37C The reactivation of the glycerol-inactivated holoenzyme and the activation of the enzymeÆCN-Cbl complex by the reactivase were assayed with 1,2-propanediol as substrate in the presence of 21 lm AdoCbl and appropriate concentrations
of ATP and MgCl2 In some experiments, ATP and MgCl2 were replaced with other nucleotides and chloride salts of divalent metal cations, respectively The protein concentra-tions of the purified enzyme and reactivase were determined
by measuring the absorbance at 280 nm, based on the method of Gill and von Hippel [34], as described previously [16]
A
B
Fig 7 Subunit swapping between DD and the reactivase (DD-R)
(A) and the existence of a cavity between DD a (pink) and b (green)
subunits (B) a, a D (pink) or a R (light blue) subunit; b, b D (green) or
bR(orange) subunit; c, cDsubunit (dark blue).