Previous studies have shown that AdoCbl-dependent methylmalonyl CoA mutase binds both coenzyme analogs in ‘base-off’ mode, which indi-cates that the histidine residue located on the cons
Trang 1adenosylcobalamin-dependent glutamate mutase
from Clostridium tetanomorphum
Hao-Ping Chen1, Huei-Ju Hsu1, Fang-Ciao Hsu1, Chien-Chen Lai2 and Chung-Hua Hsu3
1 Institute of Biotechnology, National Taipei University of Technology, Taiwan
2 Institute of Molecular Biology, National Chung-Hsing University, Taichung, Taiwan
3 Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan
Glutamate mutase from Clostridium tetanomorphum is
one of a group of adenosylcobalamin
(AdoCbl)-depen-dent mutases that catalyzes the inter-conversion of
l-glutamate and threo-b-methyl-l-aspartate It
com-prises two weakly-associating subunits, MutS and
MutE, which combine with AdoCbl to form the active
holo-enzyme [1] The coenzyme is known to be bound
by glutamate mutase in ‘base-off⁄ His-on’ mode [2] As
shown in Fig 1A, the lower axial ligand of the cobalt
atom, 5,6-dimethylbenzimidazole, is replaced by a
his-tidine residue within a conserved B12-binding motif,
DXHXXG(14–19) Model studies have shown that the
cobalt–carbon bond dissociation energy of the cofactor
is sensitive to changes in the pKa of the lower axial
base [3] This has led to speculation that proteins
might modulate the pKa of the histidine via the hydrogen bond between the His–Asp pair and so ‘fine tune’ the reactivity of AdoCbl Mutations of either residue result in significant impairment of the protein’s coenzyme-binding ability, as well as its catalytic ability [4]
The biosynthesis of AdoCbl is a very complicated process 5¢-deoxyadenosyl- cobinamide (AdoCbi) and AdoCbi-GDP are intermediates during the biosynthesis
of AdoCbl (Fig 2A) Previous studies have shown that AdoCbl-dependent methylmalonyl CoA mutase binds both coenzyme analogs in ‘base-off’ mode, which indi-cates that the histidine residue located on the conserved cobalamin-binding motif is unable to coordinate to the cobalt atom [5,6] However, the
AdoCbi-GDP-reconsti-Keywords
adenosylcobalamin; adenosylcobinamide;
AdoCbi-GDP; B 12 ; glutamate mutase
Correspondence
H.-P Chen, Institute of Biotechnology,
National Taipei University of Technology 1,
Sec 3, Chung-Hsiao East Road, Taipei 106,
Taiwan
Fax: +886 2 27317117
Tel: +886 2 27712171 ext 2528
E-mail: hpchen@ntut.edu.tw
(Received 14 August 2008, revised 30
September 2008, accepted 2 October
2008)
doi:10.1111/j.1742-4658.2008.06724.x
Adenosylcobalamin (AdoCbl)-dependent glutamate mutase from Clostrid-ium tetanomorphum comprises two weakly-associating subunits, MutS and MutE, which combine with AdoCbl to form the active holo-enzyme Three coenzyme analogs, methylcobinamide (MeCbi), adenosylcobinamide (Ado-Cbi) and adeosylcobinamide-GDP (AdoCbi-GDP), were synthesized at milligram scale Equilibrium dialysis was used to measure the binding of coenzyme B12analogs to glutamate mutase Our results show that, unlike AdoCbl-dependent methylmalonyl CoA mutase, the ratio kcat⁄ Km decreased approximately 104-fold in both cases when AdoCbi or AdoCbi-GDP was used as the cofactor The coenzyme analog-binding studies show that, in the absence of the ribonucleotide tail of AdoCbl, the enzyme’s active site cannot correctly accommodate the coenzyme analog AdoCbi The results presented here shed some light on the cobalt–carbon cleavage mechanism of B12
Abbreviations
AdoCbi, adenosylcobinamide; AdoCbl, adenosylcobalamin; Ado-PCC, (Cob-5¢-Deoxyadenosin-5¢-yl)-(p-cresyl)cobamide; (Bza)AdoCba, (benzimidazolribofuranosyl)-adenosylcobinamide; CobU, adenosyl-cobinamide kinase ⁄ adenosyl-cobinamide-phosphate guanylyltransferase; MeCbi, methylcobinamide.
Trang 2tuted enzyme is catalytically active More importantly,
the kcat⁄ Km of methylmalonyl CoA mutase is only
four-fold lower when AdoCbi-GDP is used as cofactor
[5,6] This unexpected result suggests that coordination
by the lower axial ligand is not essential in the case of
methylmalonyl CoA mutase To study the reactivity of
glutamate mutase toward these coenzyme analogs, a
chemo-enzymatic method was developed to synthesize
AdoCbi-GDP at the milligram scale Our results show
that, in contrast to methylmalonyl CoA mutase, neither
AdoCbi nor AdoCbi-GDP can efficiently act as
cofac-tor for glutamate mutase [5] The binding of AdoCbl
and three coenzyme analogs, methylcobinamide
(MeC-bi), AdoCbi and AdoCbi-GDP, to glutamate mutase
was measured by equilibrium dialysis Kinetic
proper-ties towards AdoCbi and AdoCbi-GDP were also
investigated Here, we report the results of
coenzyme-binding and kinetic studies of AdoCbl analogs with
glutamate mutase
Results
Synthesis of MeCbi, AdoCbi and AdoCbi-GDP
MeCbi and AdoCbi were successfully separated from
unreacted MeCbl and AdoCbl and the dealkylated side
products using an SP–Sepharose ion-exchange column
The relative molecular masses of MeCbi and AdoCbi
determined by ESI-MS were 1004.5 and 1240, which compare favorably with calculated relative molecular masses for MeCbi and AdoCbi of 1004.1 and 1239.6, respectively The bifunctional enzyme CobU (adenosyl-cobinamide kinase⁄ adenosyl-cobinamide-phosphate guanylyltransferase) is involved in biosynthesis and assembly of the nucleotide loop of cobalamin [7,8] (Fig 2A,B) Using chemically synthesized AdoCbi as the CobU substrate, AdoCbi-GDP was enzymatically pre-pared in large quantities The yield of AdoCbi-GDP could be significantly enhanced by using phenol⁄ dichlo-romethane extraction to remove the salt component of the AdoCbi solution The recovery of AdoCbi-GDP by reverse-phase HPLC was very reproducible (Fig 3) The relative molecular mass of AdoCbi-GDP determined by ESI-MS was 1664.4, and the calculated relative molecu-lar mass of AdoCbi-GDP is 1664.6 The HPLC method that we developed in this study is quite straightforward, separating AdoCbi and AdoCbi-GDP directly without further modification In contrast, the reactant and prod-uct, AdoCbi and AdoCbi-GDP, were analyzed in the form of (CN)2Cbi and (CN)2Cbi-GDP, respectively, in previous reports [7,8].1H-NMR spectra for MeCbi and AdoCbi have been published previously [9,10] The
600 MHz NMR spectrum of AdoCbi-GDP in
D2O⁄ H2O was analyzed using two-dimensional COSY and NOESY experiments The results are summarized
in Table 1 and Fig 2B
D14 C15
H16 G120
T121
S61
V60
L59
G92
G91
Y117
I22
L23
A118
I334
R66 A67
G68
E subunit (53.7 kDa)
S subuniT (14 kDa)
H610
D608
G609 G686
G685 G613
G653 V654 S655
Y705
T709
T706
I617
E370
E247
Q330
L374
Fig 1 (A) Model of glutamate mutase showing AdoCbl bound between the MutS and MutE subunits The coenzyme-binding domain is on the MutS subunit (B) Model of methylmalonyl CoA mutase The AdoCbl molecule is shown in grey and protein residues are shown in black.
Trang 3O NH 2
CONH 2 CONH2
H2NOC
H2NOC
CONH2
O
OH
NH
H
N N N
NH2 O
OH
HO
H H
Co
N
N
N N
AdoCbi
O NH 2
CONH2 CONH2
H2NOC
H 2 NOC
CONH 2
O
O
-P
O
HN
H
O O
N N
NNH 2 O
OH HO
H H
CoN
N
N N
N N N
N NH2 O OH O
O
-P
O
O AdoCbi-GDP
O NH 2
CONH2 CONH2
H2NOC
H 2 NOC CONH2
O NH
H3C H
N N
N NH 2 O
OH HO
H H
Co
N N
N N
AdoCbi-P P
O
O O -O
ATP
ADP
GTP
PP i
Cobinamide kinase
Cobinamide kinase
O NH 2
CONH2 CONH2
H 2 NOC
H2NOC CONH2
N O OH
HO O P O NH
H
O O
-N N
NNH 2 O
OH HO
H H
Co
N N
N N
AdoCbl
α-ribazole
GMP
Cobalamin synthase
A
Pr
AdoCbi-GDP
N N Co
NH2
O
H 2 N
H 2 N O
NH 2 O
O
NH 2
O
NH 2
O
NH
O P
O
P O O
O N
OH
N
N NH O
NH 2
R
5
3
2
1
7 8
9 10 11
12
13 14 15 16 17 18
19 20
25
26
27
30 31 32 35 37 36 38
41 42
46
47
48 49 50 53
54 55 56
57
60
61
Pr1
Pr2
3
R2
R3 R4 R5
R =
N
N N
N O OH OH
H 2 C
NH 2
A15 A13 A12 A11
O
-O
R1
A2
A8
a
b
c
d
43
e f
g
B2
A4
A5
B
Fig 2 (A) Schematic representation of the final steps of the de novo AdoCbl biosyn-thetic pathway (B) The chemical structure
of AdoCbi-GDP.
Trang 4Determination of dissociation constants for
cofactors by equilibrium dialysis
The binding of AdoCbl, MeCbi, AdoCbi and
AdoCbi-GDP to glutamate mutase was investigated by
equilib-rium dialysis Figure 4 shows the analog binding
curves with a fixed concentration of glutamate mutase
AdoCbl, MeCbi, AdoCbi and AdoCbi-GDP were
bound with apparent Kd values of 3.7 ± 0.5,
6.0 ± 0.9, 18 ± 3 and 14 ± 3 lm, respectively
(Fig 4A–D)
UV–visible spectra of protein-bound MeCbi,
AdoCbi and AdoCbi-GDP complexes
The UV–visible spectra of cobalamins provide a
useful tool to examine the coordination state of
cobalt The UV–visible absorption spectra of the
MeCbi-glutamate mutase, AdoCbi-glutamate mutase
and AdoCbi-GDP-glutamate mutase complexes were
measured A red shift was observed in the spectra of
protein-bound MeCbi, AdoCbi and AdoCbi-GDP
The 522 nm absorption maximum suggests that the
histidine residue occupies the lower axial ligand
posi-tion of the cobalt atom However, we estimate that
approximately 55–60% of the AdoCbi–glutamate
mutase complex binds the cofactor in the ‘His-on’
form (Fig 5)
150
A
B
AdoCbi
100
50
0
150
100
50
0
0 5 10 15 20
Time
25 30 35 40 45
AdoCbi-GDP
300
200
100
0
300
200
100
0
0 5 10 15 20
Time
25 30 35 40 45
Fig 3 Purification of AdoCbi-GDP from the reaction mixture
by reverse-phase HPLC (A) Before the CobU enzymatic reaction.
(B) After the CobU enzymatic reaction.
Table 1 600 MHz 1 H-NMR data for AdoCbi-GDP d, doublet; q, quadruplet; s, singlet; t, triplet; td, triplet of doublets; dd, doublet of doublets.
Assignment
Signal type
Chemical shifts AdoCbi-GDP
(p.p.m.)
J couplings (AdoCbi-GDP) (Hz) Corrin
methyl
side chain
Aminopropan-2-ol side chain
Trang 5Enzyme assay
In order to investigate the role of the ribonucleotide tail
of AdoCbl in catalysis, the coenzyme analogs were used
to examine the enzymatic activity Our results indicate
that, perhaps not surprisingly, MeCbi is a totally
inac-tive coenzyme The Km values for AdoCbi and
Ado-Cbi-GDP were 26 ± 8 and 75 ± 28 lm, respectively,
and the kcat values were (9.8 ± 1.0)· 10)3Æs)1 and
(4.5 ± 0.8)· 10)3Æs)1, respectively In both cases, the
kcat⁄ Km was decreased by approximately 104-fold
compared with that of AdoCbl
Discussion
Both methylmalonyl CoA mutase and glutamate
mutase belong to the subfamily of B12-dependent
car-bon-skeleton mutases, but their 1,2-rearrangement
mechanisms are obviously different [11] Previous
studies have shown that (a) AdoCbi does not support
the turnover of methylmalonyl CoA mutase, but
Ado-Cbi-GDP does, and (b) the enzyme binds both AdoCbi
and AdoCbi-GDP in ‘base-off⁄ His-off’ mode The
results presented here indicate that, in contrast to
methylmalonyl CoA mutase, the kcat⁄ Km of glutamate
mutase for both analogs decreased by approximately
104-fold These results suggest that the ribonucleotide
tail of AdoCbl plays an important role in catalysis in
the case of glutamate mutase In addition, both
cofac-tor analogs tested are bound by glutamate mutase in
‘base-off⁄ His-on’ mode Histidine–cobalt ligation
therefore cannot efficiently facilitate turnover of the
enzyme in the absence of the ribonucleotide tail of
AdoCbl It is apparent that glutamate mutase is
mech-anistically different from methylmalonyl CoA mutase
Significant differences in the affinity for AdoCbl between these two enzymes appear to exit Methylmal-onyl CoA mutase binds AdoCbl very tightly with a Kd
of 0.17 lm, while glutamate mutase binds AdoCbl relatively weakly with a Kd between 1.8 and 6.8 lm [1] Moreover, glutamate mutase is very sensitive to perturbation of the cofactor’s nucleotide tail, while methylmalonyl CoA mutase is not (Benzimidazolribo-furanosyl)-adenosylcobinamide [(Bza)AdoCba] is a coenzyme B12 analog in which the dimethylbenzimi-dazole moiety of AdoCbl is replaced by benzimidimethylbenzimi-dazole Previous studies have shown that the apparent Km of glutamate mutase for (Bza)AdoCba is 0.5 lm, while that for AdoCbl is 18 lm under similar conditions [12] However, the only difference between AdoCbl and (Bza)AdoCba is two methyl groups In contrast, (Co-b-5¢-Deoxyadenosin-5¢-yl)-(p-cresyl)cobamide (Ado-PCC) is another ‘base-off’ coenzyme B12 analog in which the dimethylbenzimidazole moiety of AdoCbl is replaced by a p-cresolyl group It fully supports the turnover of methylmalonyl CoA mutase The apparent
Km values of methylmalonyl CoA mutase for Ado-PCC and AdoCbl are 354 and 64 nm, respectively [13]
A structural comparison of the protein–AdoCbl com-plexes for these two enzymes is shown in Fig 1A,B The glutamate mutase-bound nucleotide tail is located
in a more crowded environment, where the space is more restricted In particular, a bulkier residue, Leu59,
is situated at the bottom of the nucleotide tail-binding pocket of glutamate mutase, but a small residue, Gly653, is located in the same position of methylmalo-nyl CoA mutase The relatively restricted space in the nucleotide tail-binding pocket might account for the low activity and affinity of glutamate mutase towards AdoCbi-GDP Our unpublished results also show that
0
0.02
0.04
0.06
0.08
0.1
0.12
A B
C D
AdoCbl (µ M )
0 0.02 0.04 0.06 0.08 0.1
0 20 40 60 80 100
MeCbi (µ M )
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0 20 40 60 80 100
AdoCbi (µ M )
0 0.01 0.02 0.03 0.04 0.05 0.06
0 20 40 60 80 100
AdoCbi-GDP (µ M )
Fig 4 Binding of AdoCbl and its analogs to glutamate mutase by equilibrium dialysis (A) AdoCbl, (B) MeCbi, (C) AdoCbi, and (D) AdoCbi-GDP The proteins, 20 l M MutE and
100 l M MutS in 0.1 mL buffer (50 m M
Tris ⁄ HCl, pH 8.5, 2 m M dithiothreitol), were dialyzed against 1 mL buffer containing
50 m M Tris ⁄ HCl, pH 8.5, 2 m M dithiothreitol and cofactors The data obtained were fitted using KALEIDA GRAPH software.
Trang 6AdoCbl-dependent lysine aminomutase binds AdoCbl
with a Kd of 18 ± 4 lm Neither AdoCbi nor
Ado-Cbi-GDP efficiently support the catalysis of
AdoCbl-dependent l-lysine or d-ornithine aminomutase [14,15]
In short, the manipulation of coenzyme B12by
methyl-malonyl CoA mutase is quite different to that by
glu-tamate mutase, l-lysine and d-ornithine aminomutase
Two mechanisms, electronic effect and steric effect,
have been postulated to explain the enzyme-accelerated
cobalt–carbon cleavage of AdoCbl [3,16] AdoCbi-GDP
is bound by methylmalonyl CoA mutase in ‘base-off’
form, and is capable of supporting the enzyme’s
cataly-sis, suggesting that the electronic effect plays a minor
role in cleavage of the cobalt–carbon bond However, as
far as we know, no experimental results from the studies
of coenzyme–protein interactions have previously been provided to support the steric effect to explain the cobalt–carbon cleavage mechanism
The binding energy for AdoCbl comes from inter-actions between proteins and the cofactor From the viewpoint of coenzyme molecule itself, these interac-tions can be divided into three parts: the ribonucleo-tide tail, corrin ring⁄ cobalt–histidine ligation, and the adenosyl group (Fig 6) As shown in Table 2, the apparent Kd values of glutamate mutase for MeCbi and AdoCbi are 6.0 ± 0.9 and 18 ± 3, respectively
As shown in Table 2, the binding energy difference between MeCbi and AdoCbi is approximately 2.5 kJÆmol)1 This result suggests that, in the absence
of the ribonucleotide tail of AdoCbl, the enzyme’s active site cannot correctly accommodate the coen-zyme analog AdoCbi In accordance with this result, the histidine residue on the conserved cobalamin-binding motif can coordinate to the cobalt atom when MeCbi is used as the cofactor (Fig 5A) How-ever, only approximately 60% of the glutamate mutase-bound AdoCbi is in the ‘base-off⁄ His-on’ form (Fig 5B) Although AdoCbi-GDP cannot effi-ciently support catalysis, its modified ribonucleotide tail helps the histidine residue coordinate to the cobalt atom (Fig 5C) Previous studies have shown that glutamate mutase binds AdoCbl, methylcobal-amin (MeCbl) and cob(II)almethylcobal-amin with similar affinity [17] These results indicate that the ribonucleotide tail of AdoCbl is important in coenzyme binding
We hereby propose that the role of the ribonucleo-tide tail of AdoCbl is to distort the adenosyl group
to fit into the enzyme’s active site during the coen-zyme-binding process However, recent spectroscopic studies have indicated that the Co–C bond of gluta-mate mutase-bound AdoCbl is not weakened within the enzyme active site [18,19] The correlation between the distortion of the adenosyl group and cleavage of the cobalt–carbon bond is still not clear Although the precise mechanism remains obscure, the results presented here do shed some light on the cobalt–carbon cleavage mechanism of B12
Experimental procedures
Materials
AdoCbl and methylcobalamin (MeCbl) were obtained from Sigma (St Louis, MO, USA) SP–Sepharose Fast Flow cat-ion-exchange gel medium was purchased from GE Health-care (Uppsala, Sweden) The production and purification of glutamate mutase from C tetanomorphum have been
0
0.1
0.2
0.3
0.4
0.5
0.6
A
B
C
350 400 450 500 550 600 650 700
Free MeCbi Protein-bound MeCbi
Wavelength (nm)
0
0.2
0.4
0.6
0.8
1
350 400 450 500 550 600 650 700
Free AdoCbi Protein-bound AdoCbi
Wavelength (nm)
0
0.05
0.1
0.15
0.2
0.25
0.3
350 400 450 500 550 600 650 700
Free AdoCbi-GDP Protein-bound AdoCbi-GDP
Wavelength (nm)
Fig 5 UV–visible spectra of free and glutamate mutase-bound
MeCbi (A), AdoCbi (B) and AdoCbi-GDP (C).
Trang 7described previously [1] All chemicals used were of
analyti-cal grade or higher
Preparation of MeCbi and AdoCbi
Because the cobalt–carbon bond of cobalamin is
light-sensitive, the following procedure was carried out in a dark
environment The chemical synthesis of AdoCbi and MeCbi
was slightly modified from that described previously [20]
For this reaction, 0.5 g of AdoCbl or MeCbl was used The
products, AdoCbi or MeCbi, were separated from the
reaction mixture using a SP–Sepharose Fast Flow
cation-exchange column (2.6· 40 cm) The column was
equili-brated in 10 mm potassium phosphate buffer, pH 7.0
AdoCbi or MeCbi were eluted with a 500 mL gradient from
0 to 0.5 m KCl The flow rate was 3 mLÆmin)1; 4 mL frac-tions were collected Fracfrac-tions containing AdoCbi or MeCbi were pooled separately The yield was approximately 30%
Chemo-enzymatic preparation of AdoCbi-GDP
The cobU gene from Salmonella typhimurium ATCC 19585 has been successfully cloned and over-expressed in Escheri-chia coli [21] CobU protein, in 50 mm Tris⁄ HCl, pH 8.5, and other solutions used for the reaction were made anaerobic and equilibrated using alternate cycles of vacuum and hydrated argon gas for 15 min The 1.5 mL reaction mixture containing 1.5 mm GTP, 1.5 mm MgCl2, 1 mm b-mercaptoethanol, 10 lm CobU and 250 lm AdoCbi was buffered in 100 mm Tris⁄ HCl, pH 8.5 Each solution was
N N H
N N
Co +3
N N H
Co+3
N
N N N
H2N
O
OH OH
OH
N N H
Co+3
OH
N N N N
H2N
O
OH OH H H
H H
Corrin ring and His ligation
Nucleotide tail
Adenosyl group
AdoCbl
No contribution
Distortion
Free energy change
contributed by:
Fig 6 Illustrations of the binding free energy change contributed by each fragment in coenzyme B 12
Table 2 Comparison of the k cat ⁄ K m value, dissociation constants and binding free energies of various coenzyme analogs The k cat ⁄ K m value for AdoCbl is calculated from the results in [1].
Coenzyme analogs Upper ligand of cobalt kcat⁄ K m (s)1Æl M )1) K
d (l M ) DG (kJÆmol)1)
Trang 8injected separately into a rubber-sealed 2 mL vial that had
been flushed with argon for 10 min prior to use The
reac-tion was incubated at room temperature overnight and was
terminated by incubation at 95C for 10 min
AdoCbi-GDP was isolated from the reaction mixture by
reverse-phase HPLC on a 5lm, 25 cm· 4.6 mm, Supelco
Ascentis C18column (Bellefonte, PA, USA) The eluents
used were as follows: eluent A, 100 mm potassium
phate buffer, pH 6.5; eluent B, 100 mm potassium
phos-phate buffer, pH 8.0 containing 50% CH3CN The flow
rate was 1 mLÆmin)1 The following profile was used for
separation: 2 min isocratic development with 98% A; 5 min
linear gradient from 98% A to 75% A; 15 min linear
gradi-ent from 75% A to 65% A; 3 min linear gradigradi-ent from
65% A to 0% A; 10 min isocratic development with 100%
B Both analogs, AdoCbi and AdoCbi-GDP, were
charac-terized by ESI-MS
NMR spectroscopy
NMR spectra of AdoCbi-GDP were recorded on a Bruker
AVANCE 600 AV system (Bruker BioSpin GmbH;
Rhein-stetten, Germany) at 25C Approximately 2 mg of
AdoCbi-GDP dissolved in 0.25 mL H2O containing 10% D2O was
used for the NMR experiment Two-dimensional
homo-nuclear (TOCSY and ROESY) and heterohomo-nuclear (HMQC
and HMBC) spectra of AdoCbi-GDP were collected for the
chemical shift assignment The ROESY spectra were
obtained with mixing times of 50 and 150 ms, to classify the
relative strengths of the observed NOEs All spectra were
pro-cessed and analyzed by using topspin 2.1 software (Bruker
BioSpin GmbH; Rheinstetten, Germany)
Measurement of the binding of coenzyme
analogs to proteins
The binding of coenzyme analogs to glutamate mutase was
measured by equilibrium dialysis About 100 lL of 20 lm
E component and 100 lm S component were loaded into
microdialysis tubes The protein solutions were dialyzed
against 1 mL of 50 mm Tris buffer, pH 8.5, in the presence
of various concentrations of coenzyme B12or its analogs at
4C overnight The absorbance was recorded at 522 nm
using an Amersham Bioscience Ultrospec 2100
spectropho-tometer; a sample of the corresponding dialysis buffer was
used to subtract the contribution of unbound coenzyme
analogs from the absorbance of the enzyme The kaleida
graph program (Synergy Software, Reading, PA, USA)
was used to fit data to estimate the dissociation constant
Protein UV–visible spectra
To determine the coordination state of the cobalt atom of
enzyme-bound coenzyme analogs, 100 lL of protein
solu-tion containing 400 lm S component, 100 lm E compo-nent, and 50 or 100 lm coenzyme analog was dialyzed against 1 mL 50 mm Tris buffer, pH 8.5, at 4C in the dark overnight, by which time equilibrium had been reached Spectra were recorded using an Amersham Bio-science Ultrospec 2100 Pro spectrophotometer (Uppsala, Sweden); a sample of the dialysis buffer was used to sub-tract the contribution of unbound coenzyme analog from the spectra of the holoenzymes
Enzyme assay
An HPLC-based method was used to assay glutamate mutase activity [22] The assay was made irreversible by coupling the formation of 3-methylaspartate to the pro-duction of mesaconate through deamination by methylas-partase In a typical reaction, 10 lm E component and
50 lm S component proteins were used in a total volume
of 100 lL containing 2 mm MgCl2, 40 mm l-glutamate and 50 mm Tris buffer, pH 8.5 The Kmand kcat for Ado-Cbi were determined in the presence of 10, 25, 50, 75 and
120 lm cofactor, and the Km and kcat for AdoCbi-GDP were determined in the presence of 20, 70, 100, 150 and
200 lm cofactor The reaction was initiated by adding
l-glutamate and incubating at room temperature for
15 min The formation of mesaconate was then analyzed
by reverse-phase HPLC on a C18 column (4.6· 250 mm)
as described previously [22]
Acknowledgements
This work was supported by grants NSC-94-2320-B-027-002 and NSC-95-2113-M-027-005-MY2 from the National Scientific Council, Taiwan, Republic of China, to H.-P.C
References
1 Holloway DE & Marsh ENG (1994) Adenosylcobala-min-dependent glutamate mutase from Clostridium tetanomorphum J Biol Chem 269, 20425–20430
2 Zelder O, Beatrix B, Kroll F & Buckel W (1995) Coordination of a histidine residue of the protein-com-ponent S to the cobalt atom in coenzyme B12-dependent glutamate mutase from Clostridium cochlearium FEBS Lett 369, 252–254
3 Halpern J (1985) Mechanisms of coenzyme B12 -depen-dent rearrangements Science 227, 869–875
4 Chen HP & Marsh ENG (1997) How enzymes control the reactivity of adenosylcobalamin: effect on coenzyme binding and catalysis of mutations in the conserved his-tidine-aspartate pair of glutamate mutase Biochemistry
36, 7884–7889
Trang 95 Chowdhury S & Banerjee R (1999) Role of the
dimeth-ylbenzimidazole tail in the reaction catalyzed by
coen-zyme B12-dependent methylmalonyl-CoA mutase
Biochemistry 38, 15287–15294
6 Chowdhury S, Thomas MG, Escalante-Semerena JC &
Banerjee R (2001) The coenzyme B12analog
5’-deoxy-adenosylcobinamide-GDP supports catalysis by
methyl-malonyl-CoA mutase in the absence of trans-ligand
coordination J Biol Chem 276, 1015–1019
7 O’Toole GA & Escalante-Semerena JC (1995)
Purifica-tion and characterizaPurifica-tion of the bifuncPurifica-tional CobU
enzyme of Salmonella typhimurium LT2 Evidence for a
CobU-GMP intermediate J Biol Chem 270, 23560–
23569
8 Thomas MG, Thompson TB, Rayment I &
Escalante-Semerena JC (2000) Analysis of the adenosylcobinamide
kinase⁄ adenosylcobinamide-phosphate
guanylyltransfer-ase (CobU) enzyme of Salmonella typhimurium LT2
Identification of residue His-46 as the site of
guanylyla-tion J Biol Chem 275, 27576–27586
9 Brown KL, Zou X & Salmin L (1991) Facile a⁄ b
dia-stereomerism in organocobalt corrins Generality of the
phenomenon and characterization of additional
a-dia-stereomers Inorg Chem 30, 1949–1953
10 Pagano TG, Yohannes PG, Hay BP, Scott JR, Finke
RG & Marzilli LG (1989) Solution behavior and
com-plete proton and carbon-13 NMR assignments of the
coenzyme B12derivative (5’-deoxyadenosyl)cobinamide
using modern 2D NMR experiments, including
600 MHz proton NMR data J Am Chem Soc 111,
1484–1491
11 Banerjee R & Rasdale SW (2003) The many faces of
vitamin B12: catalysis by cobalamin-dependent
enzymes Annu Rev Biochem 72, 209–247
12 Holloway DE, Harding SE & Marsh ENG (1996)
Adenosylcobalamin-dependent glutamate mutase:
properties of a fusion protein in which the
cobalamin-binding subunit is linked to the catalytic subunit
Biochem J 320, 825–830
13 Poppe L, Stupperich E, Hull WE, Buckel T & Retey J
(1997) A base-off analogue of coenzyme-B12with a
modified nucleotide loop1H-NMR structure analysis
and kinetic studies with (R)-methylmalonyl-CoA
mutase, glycerol dehydratase, and diol dehydratase Eur
J Biochem 250, 303–307
14 Chang CH & Frey PA (2000) Cloning, sequencing, heterologous expression, purification, and characteriza-tion of adenosylcobalamin-dependent d-lysine 5,6-ami-nomutase from Clostridium sticklandii J Biol Chem 275, 106–114
15 Chen HP, Wu SH, Lin YL, Chen CM & Tsay SS (2001) Cloning, sequencing, heterologous expression, purification and characterization of adenosylcobalamin-dependent d-ornithine aminomutase from Clostridium sticklandii J Biol Chem 276, 44744–44750
16 Pratt JM (1985) The B12-dependent isomerase enzymes; how the protein controls the active site Chem Soc Rev
14, 161–170
17 Chen HP & Marsh ENG (1997) Adenosylcobalamin-dependent glutamate mutase: examination of substrate and coenzyme binding in an engineered fusion protein possessing simplified subunit structure and kinetic prop-erties Biochemistry 36, 14939–14945
18 Brooks AJ, Fox CC, Marsh ENG, Vlasie M, Banerjee
R & Brunold TC (2005) Electronic structure studies of the adenosylcobalamin cofactor in glutamate mutase Biochemistry 44, 15167–15181
19 Sension RJ, Cole AG, Harris AD, Fox CC, Woodbury
NW, Lin S & Marsh ENG (2004) Photolysis and recombination of adenosylcobalamin bound to glutamate mutase J Am Chem Soc 126, 1598–1599
20 Hay BP & Finke RG (1987) Thermolysis of the Co–C bond in adenosylcorrins 3 Quantification of the axial base effecting adenosylcobalamin by the synthesis and thermolysis of axial base-free adenosylcobinamide Insights into the energetics of enzyme-assisted cobalt– carbon bond homolysis J Am Chem Soc 109, 8012–8018
21 Hsu FC, Ho TJ, Lai CC, Lin CF & Chen HP (2005) Cloning, sequencing, expression, and single-step purifi-cation of the adenosylcobinamide kinase⁄ adenosylcobi-namide-phosphate guanylyltransferase (CobU) from Salmonella typhimuriumATCC 19585 Protein Expr Purif 42, 178–181
22 Marsh ENG (1995) Tritium isotope effects in adenosyl-cobalamin-dependent glutamate mutase: implications for the mechanism Biochemistry 34, 7542–7547