Tài liệu Báo cáo khoa học: Cobalamin uptake and reactivation occurs through specific protein interactions in the methionine synthase–methionine synthase reductase complex docx

Cobalamin uptake and reactivation occurs through
specific protein interactions in the methionine
synthase–methionine synthase reductase complex
Kirsten R. Wolthers and Nigel S. Scrutton
Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, UK
Human methionine synthase (EC 2.1.1.13; hMS) –
an essential cellular housekeeping enzyme – produces
methionine (through the methylation of homocysteine)
and tetrahydrofolate (H
4
-folate) from the demethy-
lation of methyltetrahydrofolate (CH
3
-H
4
-folate)
(Fig. 1). Cobalamin serves as an intermediary in
methyl transfer reactions, and it cycles between the
methylcob(III)alamin and cob(I)alamin forms [1].
Cob(I)alamin is a powerful nucleophile that extracts a
relatively inert methyl group from the tertiary amine
of CH
3
-H
4
-folate. The reactive nature of cob(I)alamin
makes it susceptible to oxidation [conversion to
Keywords
chaperone; cobalamin; diflavin reductase;
methionine synthase; methionine synthase
reductase

Correspondence
N. S. Scrutton, Manchester Interdisciplinary
Biocentre and Faculty of Life Sciences,
University of Manchester, 131 Princess
Street, Manchester M1 7DN, UK
Fax: +44 161 306 8918
Tel: +44 161 306 5153
E-mail:
(Received 30 November 2008, revised 8
January 2009, accepted 21 January 2009)
doi:10.1111/j.1742-4658.2009.06919.x
Human methionine synthase reductase (MSR), a diflavin enzyme, restores
the activity of human methionine synthase through reductive methylation
of methionine synthase (MS)-bound cob(II)alamin. Recently, it was also
reported that MSR enhances uptake of cobalamin by apo-MS, a role asso-
ciated with the MSR-catalysed reduction of exogenous aquacob(III)alamin
to cob(II)alamin [Yamada K, Gravel RA, TorayaT & Matthews RG
(2006) Proc Natl Acad Sci USA 103, 9476–9481]. Here, we report the
expression and purification of human methionine synthase from Pichia
pastoris. This has enabled us to assess the ability of human MSR and two
other structurally related diflavin reductase enzymes (cytochrome P450
reductase and the reductase domain of neuronal nitric oxide synthase) to:
(a) stimulate formation of holo-MS from aquacob(III)alamin and the
apo-form of MS; and (b) reactivate the inert cob(II)alamin form of MS
that accumulates during enzyme catalysis. Of the three diflavin reductases
studied, cytochrome P450 reductase had the highest turnover rate (55.5 s
)1
)
for aquacob(III)alamin reduction, and the reductase domain of neuronal
nitric oxide synthase elicited the highest specificity (k

cat
⁄ K
m
of
1.5 · 10
5
m
)1
Æs
)1
) and MSR had the lowest K
m
(6.6 lm) for the cofactor.
Despite the ability of all three enzymes to reduce aquacob(III)alamin, only
MSR (the full-length form or the isolated FMN domain) enhanced the
uptake of cobalamin by apo-MS. MSR was also the only diflavin reductase
to reactivate the inert cob(II)alamin form of purified human MS (K
act
of
107 nm) isolated from Pichia pastoris. Our work shows that reactivation of
cob(II)alamin MS and incorporation of cobalamin into apo-MS is
enhanced through specific protein–protein interactions between the MSR
FMN domain and MS.
Abbreviations
AD, activation domain; AqCbl, aquacob(III)alamin; ATR, ATP:cobalamin adenosyltransferase; CPR, cytochrome P450 reductase; Fld,
flavodoxin; FMN
hq,
FMN hydroquinone; FMN
sq,
FMN semiquinone; FNR, FAD-dependent ferredoxin–NADP

+
reductase; H
4
-folate,
tetrahydrofolate; hMS, human methionine synthase; MeCbl, methylcob(III)alamin; MetH, cobalamin-dependent methionine synthase; MS,
methionine synthase; MSR, methionine synthase reductase; nNOSred, reductase domain of neuronal nitric oxide synthase.
1942 FEBS Journal 276 (2009) 1942–1951 ª 2009 The Authors Journal compilation ª 2009 FEBS
cob(II)alamin], an event that occurs every 200–1000
catalytic turnovers of hMS [2]. Regeneration of hMS
activity involves reductive methylation of cob(II)
alamin to form methylcob(III)alamin, a process that
couples transfer of an electron from methionine syn-
thase reductase (MSR) with methyl transfer from
AdoMet [3].
Most structural and functional information on hMS
is derived by comparison with Escherichia coli cobala-
min-dependent methionine synthase (MetH), which
shares 55% sequence identity with hMS. There are
four functional modules in hMS, arranged linearly and
separated by interdomain connectors. By analogy with
E. coli MetH, the N-terminal region of hMS comprises
two closely packed (ba)
8
barrels that bind homocyste-
ine and CH
3
-H
4
-folate [4,5]. The cobalamin-binding
module is located in the centre of the polypeptide. A

crystal structure exists for the C-terminal region of
hMS [6]. This contains the ‘activation domain’ (AD)
that binds AdoMet and MSR [7,8].
The mechanisms of reactivation of MetH and hMS
are distinct. MetH is reactivated by the transfer of
reducing equivalents from NADPH to MetH, cataly-
sed by FAD-dependent ferredoxin-NADP
+
reductase
(FNR) and mediated by flavodoxin (Fld) [2]. MSR is a
natural fusion of FNR and Fld [3,9]. It is therefore a
member of the cytochrome P450 reductase (CPR) fam-
ily [10], which also includes the reductase module of
nitric oxide synthase (nNOSred) [11,12] and a novel
oxidoreductase 1 of unknown physiological function
[13]. These proteins catalyse NADPH oxidation and
transfer electrons from the enzyme-bound FAD to the
FMN centre, and ultimately to an acceptor redox pro-
tein or domain. Although the bacterial FNR ⁄ Fld and
mammalian MSR are not interchangeable in reactivat-
ing MetH and hMS, respectively [14], human novel
oxidoreductase 1 is able to reactivate hMS, but the
functional significance of this is unknown [15].
In addition to electron transfer activity, MSR also
has putative chaperone-like activity; it promotes the
stability of hMS by facilitating uptake of cobalamin
by the apo-form of hMS [14]. The enhanced cofactor
binding is thought to result from MSR-catalysed
reduction of exogenous aquacob(III)alamin (AqCbl) to
form cob(II)alamin. Reduction of the Co centre

promotes the dissociation of the lower dimethylbenz-
imidazole base of the cofactor. Consistent with this,
the crystal structure of the cobalamin-binding domain
Homocysteine
Methionine
Co
Methylcob(III)alamin
Cob(I)alamin
Primary
turnover
cycle
H
CH
3
H
4
folate
AdoHyc
Cob(I)alamin
Cob(II)alamin
H
4
folate
Co
NADPH
A
doMet
e
–
FAD

FMN
NADPH
NADP
+
e
–
Reactivation
Co
Fig. 1. Catalytic scheme and proposed conformational states of hMS during primary turnover and reactivation. hMS transfers a methyl group
from methylcob(III)alamin to homocysteine, generating cob(I)alamin and methionine. A methyl group is then abstracted by cob(I)alamin from
CH
3
-H
4
-folate, generating H
4
-folate and the methylcob(III)alamin form of MS. During primary turnover, the homocysteine-binding domain (dot-
ted barrel) and the CH
3
-H
4
-folate binding-domain (black barrel) form discrete complexes with the cobalamin-binding domain (dark grey circle).
hMS is inactivated approximately every 200–1000 catalytic turnovers [owing to the highly reactive nature of cob(I)alamin], to yield the inert
cob(II)alamin form of hMS. Reductive methylation of cob(II)alamin, a process involving electron transfer from MSR and methyl transfer from
S-adenosylmethionine, regenerates the active form of hMS. During reactivation of hMS, the FMN domain of MSR (light grey) and the C-ter-
minal activation of hMS (grid-barrel) interact with the cobalamin-binding domain. For more information on hMS conformational substates,
see [5] and [33].
K. R. Wolthers and N. S. Scrutton Formation of holo-methionine synthase
FEBS Journal 276 (2009) 1942–1951 ª 2009 The Authors Journal compilation ª 2009 FEBS 1943
of MetH reveals that the dimethylbenzimidazole base

is buried within the protein scaffold, well removed
from the corrin ring, suggesting that the lower-coordi-
nated Co state preferentially binds to hMS [7].
Herein, we report for the first time the development
of an expression and purification system for hMS
based on the expression host Pichia pastoris. This has
enabled us to investigate: (a) the potential for cobala-
min incorporation mediated by other mammalian difla-
vin reductases and also subdomains of MSR; (b) the
extent of reductive remethylation of hMS catalysed by
the different redox states of MSR; and (c) the ability
of structurally related diflavin reducatases to reactivate
hMS. These studies have enabled us to refine the chap-
erone-like role of MSR. We show that specific pro-
tein–protein interactions between hMS and MSR (over
and above the need to catalyse the reductive chemistry)
are required to promote the insertion of the cobalamin
into hMS. We also demonstrate that the chaperone-
like role is orchestrated entirely through the FMN
domain of MSR and is not linked to MSR-catalysed
reduction of exogenous AqCbl to form cob(II)alamin
as previously proposed [14].
Results and Discussion
Purification of hMS
The ability to express hMS in a recombinant and func-
tional form has been a major limitation in studies of
the hMS and MSR redox system. However, we found
that recombinant hMS is expressed as an apoenzyme
in P. pastoris at levels that enable purification of suffi-
cient quantities for functional analysis (Table 1). A

clear advantage of using Pichia as a heterologous host,
as opposed to other eukaryotic expression systems, is
the capacity to grow large-scale cultures on relatively
inexpensive media. The fact that the enzyme is
expressed in the apo-form is consistent with yeast
being unable to synthesize cobalamin or transport it
across the cell membrane [16]. We purified hMS using
two steps, employing ion exchange chromatography
followed by cobalamin affinity chromatography
(Table 1). The affinity chromatography step conve-
niently converts the apo-form of hMS into the holoen-
zyme. The activity of hMS through all purification
steps was determined using a nonradioactive spectro-
photometric assay (see Experimental procedures).
Recombinant hMS was found to be homogeneous
after cobalamin affinity chromatography, as judged by
SDS ⁄ PAGE analysis (Fig. 2, inset). The absorption
spectrum of the purified enzyme was typical of the
hydroxycobalamin form of the enzyme (Fig. 2). The
recovery of the activity was  10%, and the enzyme
was purified 3669-fold. The specific activity and yield
of purified hMS were similar to the values obtained
using the baculovirus expression system [14].
Reactivation of hMS by MSR
Reductive activation of hMS by MSR was measured
by following the incorporation of
14
CH
3
into methio-

Table 1. Purification of hMS from an expressing strain of P. pastoris. The crude extract was generated from 103 g of wet Pichia pastoris
cell pellet containing the integrated pPICZMS plasmid. hMS activity was measured using the discontinuous spectroscopic assay outlined in
Experimental procedures.
Total protein
(mg)
Total activity
(nmolÆmin
)1
)
Specific activity
(nmolÆmin
)1
Æmg
)1
) Yield (%)
Purification
n-fold
Crude extract 8500 4100 0.5 100
Q-Sepharose eluate 1880 2100 1.1 50 2.3
Cobalamin eluate 0.3 420 1540 10 3669
Fig. 2. UV–visible spectrum of hMS following elution of enzyme
from cobalamin–agarose resin. Inset: SDS polyacrylamide gel (8%)
analysis indicating the purity of hMS recovered from the cobala-
min–agarose resin. Protein was visualized by staining with
Coomassie Brilliant Blue R250. Lane 1: protein markers (200, 116,
97, 66 and 45 kDa). Lane 2: purified hMS.
Formation of holo-methionine synthase K. R. Wolthers and N. S. Scrutton
1944 FEBS Journal 276 (2009) 1942–1951 ª 2009 The Authors Journal compilation ª 2009 FEBS
nine from
14

CH
3
-H
4
-folate. The rate of
14
CH
3
incorpo-
ration was found to saturate with respect to MSR
concentration (Fig. 3A). The parameter K
act
defines
the MSR concentration that defines 0.5 of the total
recoverable activity of hMS, and was calculated to be
107 ± 14 nm; the maximal recoverable activity at satu-
ration (k
cat
) was 1.5 lmolÆmin
)1
Æmg
)1
, which is similar
to previously reported values for nonrecombinant
forms of hMS [3,14]. Reactivation of hMS was not
observed when MSR was replaced by nNOSred or
CPR, highlighting the need for specific protein–protein
interactions between MSR and hMS. Reactivation of
hMS was found to be dependent on NADPH concen-
tration in a hyperbolic manner (Fig. 3B), yielding an

apparent K
m
for NADPH of 23.2 ± 3.4 lm. This
value is approximately 10-fold higher than that
reported previously for purified porcine methionine
synthase (MS), but we emphasize that studies with the
porcine enzyme were conducted under different assay
conditions [3]. Previously, we measured, by isothermal
thermal calorimetry and product inhibition studies, an
apparent K
d
of 37 lm for the MSR–NADP
+
complex
[17], and, by stopped-flow experiments, an apparent K
d
of  50 lm for NADPH for the MSR–NADPH com-
plex [18]. These values are in reasonable agreement
with the apparent K
m
for NADPH measured in our
reactivation assays.
Reactivation of hMS by different redox states of
MSR and the isolated FMN domain
There is a significant thermodynamic barrier to elec-
tron transfer from either the MSR FMN semiquinone
(FMN
sq
) or FMN hydroquinone (FMN
hq

) to the
hMS-bound cob(II)alamin [19]. Specifically, the mid-
point potential values for FMN
ox ⁄ sq
and FMN
sq ⁄ hq
are respectively 380 and 270 mV more electropositive
than the putative midpoint potential of the cob(II)
alamin ⁄ cob(I)alamin couple (determined for MetH
[20]), which equates to free energy changes of 36 and
26 kJÆmol
)1
, respectively [21–23], for electron transfer
between the two cofactors.
We examined whether reductive methylation of
hMS–cob(II)alamin requires full or partial reduction
of MSR [i.e. whether electron transfer to cob(II)alamin
occurs from FMN
sq
or FMN
hq
]. We reduced MSR or
the isolated FMN domain under anaerobic conditions
by titration with dithionite to the desired redox state,
and then mixed prereduced enzyme with the remaining
reaction components (see Experimental procedures). In
an anaerobic reaction mixture, hMS was able to cata-
lytically turn over in the absence of a reactivation
partner (Table 2). This is at first sight a puzzling result,
as hMS was isolated in the inactive form with the co-

factor in the Co
3+
oxidation state (i.e. AqCbl). Activity
may arise from: (a) a small amount of enzyme present in
the active methylcob(III)alamin (MeCbl) form; or (b) the
presence of a reducing agent (e.g. thiols) converting
A
B
Fig. 3. (A) The dependence of hMS activity on MSR concentration,
illustrating the optimal concentration of MSR required for reductive
methylation (reactivation) of the cob(II)alamin form of hMS. hMS
assays were performed using the radioactive assay described in
Experimental procedures, in which AqCbl and dithiothreitol were
replaced with varying concentrations of MSR and 100 l
M NADPH.
The experimental data were fitted to a hyperbolic equation, and
yielded a K
act
value for MSR of 107 ± 1 nM and a V
max
value of
2.0 ± 0.1 nmolÆmin
)1
. (B) The dependence of hMS activity on
NADPH concentration, illustrating the optimal concentration of
NADPH required for reductive methylation (reactivation) of the
cob(II)alamin form of hMS. hMS assays were performed as
described in Experimental procedures, using an MSR concentration
of 6 l
M. The experimental data for NADPH were fitted to a hyper-

bolic equation, yielding a K
m
for NADPH of 23.2 ± 3.4 lM.
K. R. Wolthers and N. S. Scrutton Formation of holo-methionine synthase
FEBS Journal 276 (2009) 1942–1951 ª 2009 The Authors Journal compilation ª 2009 FEBS 1945
AqCbl to cob(II ⁄ I)alamin, an event that is more feasi-
ble in an anaerobic environment [24]. We found that
the addition of oxidized FMN domain to the reaction
mixture resulted in an  3-fold increase in hMS turn-
over, despite the FMN cofactor being preoxidized by
FeCN. The presence of a reducing agent (e.g. natural
light or thiols; see Table 2 footnote) in the reaction
mixture may reduce a proportion of the FMN domain,
converting some of the enzyme to the active form. The
 3-fold increase in activity may arise from the binding
of the FMN domain to hMS facilitating binding of
AdoMet and ⁄ or methyl transfer, although this has not
been formally shown. It is known that the isolated
FMN domain in the oxidized form does bind to the
hMS AD [19]. We found that FMN
sq
and FMN
hq
increased hMS product yield by 8- and 11-fold, respec-
tively. This indicates that the isolated FMN domain
participates in the reductive remethylation of hMS,
which by necessity involves endergonic electron transfer
from FMN
sq
to cobalamin. This energetically unfa-

vourable electron transfer is tightly coupled to methyl
group transfer from AdoMet (a highly exothermic reac-
tion), which drives the net reaction forwards.
Oxidized MSR does not reactivate hMS (Table 2).
In fact, the activity of hMS was found to be less than
in the absence of any flavoprotein, which may be
attributed to a tendency of oxidized MSR to withdraw
reducing equivalents from hMS, thereby inhibiting the
reactivation process. Alternatively, the binding of oxi-
dized MSR to hMS may prevent an exogenous reduc-
ing agent from reducing hMS and returning it to the
catalytic cycle. The turnover of hMS increases as MSR
is reduced to the one-electron, two-electron and four-
electron reduced states, reflecting a higher concentra-
tion of reducing equivalents needed to return hMS to
the catalytic cycle.
Reduction of free AqCbl by diflavin reductases
MSR was shown previously to reduce AqCbl to
cob(II)alamin, and this activity is thought to facilitate
the uptake of cobalamin by the apo-form of hMS [14].
We studied the ability of other diflavin reductase
enzymes to reduce AqCbl and facilitate uptake of
cobalamin by apo-MS. We found that CPR, nNOSred
and MSR reduced AqCbl to cob(II)alamin (Table 3).
CPR has the highest turnover number for NADPH-
catalysed reduction of AqCbl (55.5 s
)1
), > 20-fold
that of MSR (2.7 s
)1

) and  6-fold that of nNOSred
(9.0 s
)1
). Calculated values for specificity constants
(k
cat
⁄ K
m
) reveal that CPR has the greatest specificity
(10.6 · 10
5
m
)1
Æs
)1
) for AqCbl, with MSR
(4.1 · 10
5
m
)1
Æs
)1
) and nNOSred (1.5 · 10
5
m
)1
Æs
)1
)
working less effectively with this substrate. We demon-

strated that the AqCbl reductase activity is dependent
on the FMN domain, because the isolated NADP(H) ⁄
FAD domain of MSR was unable to reduce this cofac-
tor directly. Thus, AqCbl can be likened to cyto-
chrome c
3+
, in that it serves as a nonphysiological
electron acceptor of diflavin reductases, but in doing
so it takes electrons only from the FMN domain.
Table 2. Anaerobic reactivation of hMS by different redox forms of
MSR and the isolated MSR FMN domain. Various redox forms of
MSR, or the FMN domain (40 l
M), were added to an assay mixture
containing 0.2
M potassium phosphate buffer (pH 7.2), 100 lM Ado-
Met, 1 m
M homocysteine, 250 lM
14
CH
3
-H
4
-folate (1200 d.p.m. per
nmol), and hMS, in a total volume of 250 lL. The reaction was
incubated for 10 min at 37 °C, and quenched and analysed follow-
ing the protocol for the radioactive hMS activity assay described in
Experimental procedures.
Redox form MS activity (nmolÆmin
)1
)

Control – no hMS < 0.01
Without flavoprotein
a
0.24 ± 0.02
FMN domain oxidized
b
0.66 ± 0.05
FMN domain 1
e)
1.86 ± 0.17
FMN domain 2
e)
2.65 ± 0.18
MSR oxidized 0.03 ± 0.02
MSR 1e
)
1.11 ± 0.11
MSR 2e
)
1.19 ± 0.09
MSR 4e
)
1.61 ± 0.11
a
The low level of hMS activity seen in the absence of flavoprotein
may arise from a small fraction of hMS in the MeCbl form following
purification.
b
The increase in hMS activity shown in the presence
of the oxidized FMN domain may be due to photoreduction of the

FMN cofactor by natural light, in particular during gel filtration to
remove excess FeCN [34]. Alternatively, a small amount of reduc-
ing agent (e.g. thiols) present in the assay may be reducing FMN
and ⁄ or the cobalamin. The source of the reducing agent is
unknown, but it may originate from dithionite on the surface of the
gloves in the anaerobic glove box or surface-exposed thiols on the
proteins themselves.
Table 3. Kinetic parameters obtained for the NADPH-catalysed
reduction of AqCbl to cob(II)alamin. The rate of NADPH-catalysed
reduction of AqCbl by MSR, CPR and nNOSred was measured by
following the decrease in absorbance at 525 nm. Reactions were
performed in 50 m
M potassium phosphate buffer (pH 7.2), 85 lM
NADPH, 5–20 · 10
12
mol of enzyme, and variable concentrations of
AqCbl, in a total volume of 1 mL, at 37 °C.
Enzyme k
cat
(s
)1
) K
m
(lM)
k
cat
⁄ K
m
(· 10
5

M
)1
Æs
)1
)
MSR 2.7 ± 0.1 6.6 ± 0.4 4.1 ± 0.3
CPR 55.5 ± 1.3 52.4 ± 2.7 10.6 ± 0.6
nNOSred 9.0 ± 0.3 60.2 ± 4.0 1.5 ± 0.1
Formation of holo-methionine synthase K. R. Wolthers and N. S. Scrutton
1946 FEBS Journal 276 (2009) 1942–1951 ª 2009 The Authors Journal compilation ª 2009 FEBS
Holoenzyme synthase activity of diflavin
reductases
The fact that CPR and nNOSred can enzymatically
reduce AqCbl to cob(II)alamin prompted us to investi-
gate whether these reductases can mimic MSR [14] by
enhancing the uptake of cobalamin by apo-MS. It was
previously shown that apo-MS generated by expression
in insect cells or purified from rat liver is unstable at
37 °C [14,25]. In our studies, hMS activity dropped to
0.01 nmolÆmin
)1
in Pichia cell extracts expressing apo-
MS which were incubated at 37 °C for 70 min in the
absence of cobalamin (Table 4). The addition of MSR,
nNOSred or CPR to samples for which cobalamin was
omitted had a negligible affect on activity (0.02–
0.04 nmolÆmin
)1
; data not shown). The addition of
AqCbl or MeCbl to the crude extract resulted in a

small increase in activity ( 13 and  6-fold, respec-
tively), which was not greatly affected by the addition
of CPR or nNOSred. In contrast, the presence of
MSR along with AqCbl or MeCbl caused a dramatic
increase in hMS activity (7-fold for AqCbl and 20-fold
for MeCbl) as compared to the cofactor alone. Simi-
larly, the addition of the FMN domain of MSR along
with AqCbl and MeCbl caused a 6-fold and 20-fold
stimulation of hMS activity. The presence of NADPH
in the preincubation mixture along with MSR and
MeCbl ⁄ AqCbl did not have a significant effect in stim-
ulating hMS activity.
These results indicate that although uptake of cobal-
amin by apo-MS is not entirely dependent on MSR,
the enzyme does greatly enhance the stability of the
apoenzyme. During purification of hMS from Pichia,
we measured hMS activity in crude extract by the
AqCbl ⁄ dithiothreitol assay (see Experimental proce-
dures), which does not contain MSR. Thus, the cofac-
tor can be incorporated into apo-MS by a diffusive
process. However, it is clear from Table 4 that MSR
enhances the stability of apo-MS, and that this stabil-
ization effect is strictly dependent on the presence of
AqCbl or MeCbl. It is possible to infer from these
data that MSR is eliciting a ‘holosynthase-like’ func-
tion. Our studies show that the mechanism by which
MSR serves as a putative molecular chaperone for
hMS does not rely on NADPH-catalysed reduction of
exogenous AqCbl to cob(II)alamin: this follows
because (a) hMS activity is not stimulated by the addi-

tion of NADPH to the preincubation mixture, (b) the
FMN domain has a similar effect to that of full-length
MSR in improving hMS stability, (c) MSR enhances
hMS stability with both AqCbl and MeCbl, and (d)
CPR and nNOSred are unable to affect hMS stability,
despite having AqCbl reductase activity. Therefore, the
incorporation of cobalamin mediated by MSR requires
specific interaction between MSR and hMS, and in
particular contact through the FMN domain, analo-
gous to that for the hMS–MSR reactivation complex.
The sequestering of cobalamin between two partner
proteins has been observed for in vitro formation of
adenosylcobalamin by MSR and ATP:cobalamin ade-
nosyltransferase (ATR) [26]. In this system, MSR and
ATR form a complex to sequester the highly reactive
cob(I)alamin intermediate that is formed in the MSR-
catalysed reduction of cob(II)alamin. The containment
of the B
12
cofactor within a protein complex poten-
tially facilitates effective adenosylation of cob(I)alamin
by ATR to form adenosylcobalamin.
Previously, we have shown that the addition of the
hMS AD to the FMN domain or full-length MSR
results in a quenching of the intrinsic flavin fluores-
cence, suggesting that the flavin chromophore is
shielded from the solvent in the protein–protein com-
plex [19]. From the fluorescence titration assays, an
apparent dissociation constant (K
d

of 4.5 lm) for the
complex was determined for the hMS AD–MSR
complex, which closely mimics that of the MetH–Fld
system (see Fig. S1 and Doc. S1) [22]. Titration of
CPR or nNOSred with the hMS AD did not result in
Table 4. Holoenzyme synthase (chaperone) activity of diflavin
reductases. Crude extract (150 lL) of P. pastoris expressing recom-
binant apo-MS was preincubated at 37 °C for 70 min, with and
without the components indicated, in a total volume of 200 lL. Fol-
lowing incubation, the activity of hMS was measured by the radio-
active assay described in Experimental procedures, using the
AqCbl ⁄ dithiothreitol reducing system. The concentrations of the
components were 200 n
M MSR, MSR FMN domain, CPR or nNOS-
red, 50 l
M MeCbl or AqCbl, and 200 lM NADPH.
Apo-MS treatment
hMS activity (nmolÆmin
)1
)
70 min
No Cbl
Without NADPH or MSR 0.01 ± 0.01
With MSR 0.02 ± 0.01
With NADPH and MSR 0.02 ± 0.01
AqCbl
Without MSR or NADPH 0.13 ± 0.01
With MSR 0.91 ± 0.14
With FMN domain 0.75 ± 0.02
With NADPH and MSR 1.12 ± 0.04

With NADPH and CPR 0.09 ± 0.02
With NADPH and nNOSred 0.18 ± 0.02
MeCbl
Without MSR or NADPH 0.06 ± 0.01
With MSR 1.23 ± 0.15
With FMN domain 1.20 ± 0.10
With NADPH and MSR 1.19 ± 0.03
With NADPH and CPR 0.20 ± 0.01
with NADPH and nNOSred 0.19 ± 0.02
K. R. Wolthers and N. S. Scrutton Formation of holo-methionine synthase
FEBS Journal 276 (2009) 1942–1951 ª 2009 The Authors Journal compilation ª 2009 FEBS 1947
a quenching of flavin fluorescence, confirming that
these two proteins do not interact with hMS. We have
compared the electrostatic potentials for the surface of
the CPR FMN domain (in the region of the solvent-
exposed FMN) with that of a homology model of the
MSR FMN domain (based on the structure of the
CPR FMN domain; Protein Data Bank: 1b1c) (see
Doc. S1 and Fig. S2). The surface corresponding to
the binding region for hMS AD is considerably less
negatively charged in MSR than in the corresponding
region of CPR. The electrostatic surface potential of
the hMS AD (Protein Data Bank code: 202K) also
contains relatively few charged groups near the
S-adenosylmethionine-binding site (see Fig. S3 and
Doc. S1). Thus, the propensity of hydrophobic resi-
dues on the putative binding interface of the MSR
FMN domain suggests less emphasis on electrostatic
interactions mediating hMS–MSR complex formation
as compared to that of the CPR–P450 redox pair.

In conclusion, both FMN
sq
and FMN
hq
of MSR
can participate in reductive methylation of hMS. MSR
has a second important physiological function in facili-
tating uptake of cobalamin by hMS, a role that neces-
sitates formation of an hMS–MSR complex. The latter
finding is potentially important for future investiga-
tions into how polymorphic or clinical mutants of
MSR manifest in disease states, such as hyperhomo-
cysteinemia or megaloblastic anemia.
Experimental procedures
Reagents
Hydroxycobalamin, MeCbl, AdoMet and homocysteine
thiolactone were obtained from Sigma Chemical Company
(Poole, UK). Restriction enzymes and T4 DNA ligase were
from New England Biolabs (Hitchin, UK). Pfu Turbo
DNA polymerase and XL1-blue competent cells were
purchased from Stratagene (La Jolla, CA, USA). CH
3
-
H
4
-folate and 5-[
14
C]methyl-H
4
-folate were obtained from

Schircks Laboratories (Jona, Switzerland) and Amersham
Biosciences UK Ltd (Chalfont St Giles, UK), respectively.
Oligonucleotides were supplied by Invitrogen (Paisley, UK).
Heterologous expression of hMS in P. pastoris
The cloning and mutagenesis of the cDNA for hMS is
described in Doc. S1. The sequences of the oligonucleotides
used for cloning and mutagenesis of the hMS cDNA are
listed in Tables S1–S2. The pPICZMS plasmid was digested
with Pme1, and the linearized plasmid was transformed into
P. pastoris strain SMD1168 by electrophoration, using the
protocol outlined in the manual supplied by the commercial
supplier of the strain (Invitrogen). Transformed colonies
were selected on YPDS [1% (w ⁄ v) yeast extract, 2% (w ⁄ v)
peptone, 1 m sorbitol, 2% (w ⁄ v) dextrose] plates containing
100 lgÆmL
)1
zeocin. Several transformed colonies were
streaked onto plates containing 1000 lgÆmL
)1
zeocin to
select for colonies containing multiple copies of the inte-
grated cDNA for hMS. The fermentative growth of Pichia
was adapted from the Invitrogen protocol (Pichia Fermenta-
tion Growth Guidelines; Invitrogen). Expression of recom-
binant hMS was obtained by first inoculating 5 mL of
BMGY medium [1% (w ⁄ v) yeast extract, 0.5% (w ⁄ v) pep-
tone, 100 mm potassium phosphate, pH 6.0, 1.34% yeast
nitrogen base, 0.4 lgÆmL
)1
biotin, and 1% (w ⁄ v) glycerol]

with a single transformed colony, and incubating the culture
for 8 h at 30 °C with gentle aeration. The 5 mL culture was
then used to inoculate 200 mL of BMGY medium, which
was subsequently incubated for 16 h at 30 °C with gentle
aeration. Fermentation of the Pichia culture was performed
in a 7.5 L Bioflo 110 benchtop fermenter (New Brunswick
Scientific, Edison, NJ, USA) equipped with microprocessor
control of pH, dissolved oxygen, agitation, temperature,
and nutrient feed, and with electronic foam control. The
vessel contained 3.5 L of media comprising 0.93 gÆL
)1
CaSO
4
, 18.2 gÆL
)1
K
2
SO
4
, 14.9 gÆL
)1
MgSO
4
.7H
2
O,
26.7 mL of 85% phosphoric acid, 4.13 g of KOH, and
40 gÆL
)1
glycerol, along with 4.25 mLÆL

)1
of trace salts
(PTM
1
; Invitrogen). The fermentation medium was inocu-
lated with 200 mL of starter culture. Throughout growth,
the temperature was maintained at 29 °C, and agitation was
constant at 900 r.p.m. A pH of 5.0 was maintained using
14% (w ⁄ v) ammonium hydroxide. The glycerol batch phase
was run until glycerol was completely consumed ( 22 h).
During the second phase of growth (the ‘methanol–glycerol
mix feed phase’), glycerol (containing 12 mL of PTM
1
trace
salts per litre) was added to the culture at 3.6 mLÆh
)1
ÆL
)1
of
initial fermentation volume. After 1 h, methanol (containing
12 mL of PTM
1
trace salts per litre) was added to the cul-
ture at 1.2 mLÆh
)1
ÆL
)1
of initial fermentation volume. After
an additional 1 h, the methanol flow rate was increased to
2.4 mLÆh

)1
ÆL
)1
and the glycerol feed rate was decreased to
2.4 mLÆh
)1
ÆL
)1
. Finally, after another 2 h, the methanol
flow rate was increased to 3.6 mLÆh
)1
ÆL
)1
of initial fermen-
tation volume, and the glycerol feed was terminated. The
cells continued to grow on methanol supplied at 3.6 mLÆ
h
)1
ÆL
)1
of initial fermentation volume for a further 24 h.
The cells were centrifuged at 3000 g for 10 min, and the wet
cell pellet was frozen at )80 °C.
Purification of hMS
Human MS was purified by ion exchange and cobalamin
affinity chromatography, following a modified protocol of
Yamada et al. [14]. Cobalamin–agarose was prepared
according to the method of Sato et al. [27]. All purification
steps were performed on ice or at 4 °C, unless otherwise
Formation of holo-methionine synthase K. R. Wolthers and N. S. Scrutton

1948 FEBS Journal 276 (2009) 1942–1951 ª 2009 The Authors Journal compilation ª 2009 FEBS
stated. Cells (103 g wet weight) were suspended in 250 mL of
50 mm potassium phosphate buffer (pH 7.2), containing
1mm phenylmethanesulfonyl fluoride and two protease
inhibitor tablets (Roche Products Ltd, Welwyn Garden City,
UK). The cells were disrupted by passing the cell suspension
twice through a cell disrupter (T-series Cabinet; Constant
Systems, Daventry, UK) at 40 000 lb in
)2
. The cell debris
was centrifuged at 40 000 g for 45 min. The supernatant was
applied to a Q-Sepharose Fast Flow column (5 · 11 cm)
equilibrated with 50 mm potassium phosphate buffer
(pH 7.2). The protein was eluted with a linear gradient
(0–0.5 m NaCl) at 2 mLÆmin
)1
. Fractions containing hMS
activity were pooled and mixed with the cobalamin–agarose
(12 mL) at 22 °C for 1 h. The mixture was then packed into
a column (1.5 cm in diameter). The resin was washed with
50 mm potassium phosphate buffer (pH 7.2), followed by
50 mm potassium phosphate buffer (pH 7.2) containing 1 m
NaCl, and then equilibrated in 10 mm Tris ⁄ HCl (pH 7.2).
The resin, as a 50% slurry, was placed into a 25-mL beaker
and exposed to light (halogen lamp; SCHOOT-KL1500 LCD
set at 3300 K for 15 min on ice). The slurry was then loaded
into a column (1.5 cm in diameter), and the resin was washed
with 10 mm Tris ⁄ HCl (pH 7.2). Human MS was eluted with
10 mm Tris ⁄ HCl (pH 7.2) containing 0.5 m NaCl, and then
dialysed against 50 mm potassium phosphate buffer (pH 7.2)

for 16 h. Purified hMS was concentrated and stored at
)80 °C. Protein concentrations at various purification steps
were determined using the Bio-Rad (Hercules, CA, USA)
protein assay kit, using BSA as a standard. Purification of
human CPR [28], rat nNOSred [29] and human MSR [21]
followed previously published protocols.
Nonradioactive hMS activity assay
The activity of hMS during purification was measured
using a discontinuous spectrophotometric assay in which
the product CH
3
-H
4
-folate is converted to CH
+
=H
4
-folate
[30]. The reaction mixture contained 0.1 m potassium phos-
phate buffer (pH 7.2), 0.1 m KCl, 250 l m CH
3
-H
4
-folate,
1mm homocysteine, 100 lm AdoMet, 25 mm dithiothreitol,
and 50 lm AqCbl. The total reaction volume was 0.8 mL.
The reactions were initiated by the addition of homo-
cysteine following 5 min of incubation at 37 °C of the
enzyme with all other components. Following 10 min of
incubation at 37 °C, the reaction was quenched by the

addition of 0.2 mL of a solution containing 11 m formic
acid and 5 m HCl, and heated to 90 °C for 10 min. The
acidification of the reaction mixture quantitatively converts
CH
3
-H
4
-folate to CH
+
=H
4
-folate, which absorbs strongly
at 350 nm (De =26500m
)1
cm
)1
).
Radioactive hMS activity assay
A radioactive hMS assay that monitors the transfer of the
[
14
C]methyl group from CH
3
-H
4
-folate to the product
methionine was adapted from a published protocol [14].
The assay mixture comprised 0.2 m potassium phosphate
buffer (pH 7.2), 100 lm AdoMet, 1 mm homocysteine,
50 lm AqCbl, 25 mm dithiothreitol, and 250 lm

14
CH
3
-H
4
-
folate (1200 d.p.m. per nmol), in a total volume of 250 lL.
All the components were mixed and incubated at 37 °C for
3 min. The reaction was initiated by the addition of 1 mm
homocysteine, and then further incubated at 37 ° C for
10 min. The reaction was quenched at 90 °C for 3 min, and
the reaction mixture was then cooled to room temperature
before being applied to a 2 mL (0.8 · 4 cm) AG-1 (Bio-
Rad) column. The column was washed with 2 · 1mL of
water. The combined radioactivity in the flowthrough and
wash fractions was quantitated by scintillation counting.
For MSR-catalysed assays (used for measuring the K
m
of
NADPH and the K
act
of MSR), AqCbl and dithiothreitol
were replaced by varying concentrations of MSR and
NADPH.
Anaerobic hMS reactivation assay
The anaerobic hMS assays were performed in a Belle Tech-
nology glove box (O
2
> 5 p.p.m.) equipped with an Hita-
tchi U-1800 spectrophotometer. All buffers and reaction

mixtures were extensively bubbled with nitrogen prior to
introduction into the glove box. A concentrated sample of
MSR or the isolated FMN domain was introduced into the
glove box, and FeCN was added to the concentrated pro-
tein stock to fully oxidize the flavin cofactors. To remove
excess FeCN and O
2
, MSR and the isolated FMN domain
were gel filtered using a 10 mL Econo-pack column (Bio-
Rad) equilibrated with anaerobic buffer (10 mm potassium
phosphate, pH 7.2). The enzymes were reduced to the 1, 2
and 4 (in the case of full-length MSR) reduced states by
titration with dithionite. The UV–visible spectra of the
flavoproteins were recorded with sequential addition of
dithionite. The various reduced forms of MSR, or the
FMN domain (40 lm), were added to an assay mixture
containing 0.2 m potassium phosphate buffer (pH 7.2),
100 lm AdoMet, 1 mm homocysteine, 250 l m
14
CH
3
-H
4
-
folate (1200 d.p.m. per nmol), and hMS, in a total volume
of 250 lL. The reaction was incubated for 10 min at 37 °C,
and quenched and analysed following the protocol for the
radioactive hMS activity assay. The concentration of MSR
and the FMN domain were determined by the absorbance
value at 450 nm, using extinction coefficients of 25 600 and

14 700 m
)1
Æcm
)1
, respectively [21].
Measurement of cobalamin reductase activity
The rates of NADPH-catalysed reduction of AqCbl by
MSR, human CPR and nNOSred were measured by follow-
ing the decrease in absorbance at 525 nm using a difference
extinction coefficient of 5.57 · 10
)3
m
)1
Æcm
)1
[31] on a
Cary50 spectrophotometer. Reactions were performed in
K. R. Wolthers and N. S. Scrutton Formation of holo-methionine synthase
FEBS Journal 276 (2009) 1942–1951 ª 2009 The Authors Journal compilation ª 2009 FEBS 1949
50 mm potassium phosphate buffer (pH 7.2), 85 lm
NADPH and variable concentrations of AqCbl, at 37 °C,
in a total volume of 1 mL. The reaction was initiated by
adding 5–20 · 10
12
mol of enzyme. The concentrations of
CPR and nNOSred were determined by the absorbance
value at 450 nm, using extinction coefficients of 22 000 and
21 600 m
)1
Æcm

)1
, respectively [29,32].
Measurement of holo-MS synthase activity
Holo-MS synthase activity was measured following a previ-
ously published protocol [14]. Pichia cells expressing recom-
binant hMS were disrupted, and the crude extract (2 mL)
was applied to a 10 mL gel filtration column to remove
small molecules. The filtered extract was then incubated for
70 min at 37 °C in the presence or absence (as noted) of
NADPH, MSR, FMN domain of MSR, CPR, nNOSred,
AqCbl and MeCbl. The activity of the holo-MS was then
measured by the AqCbl ⁄ dithiothreitol radioactive assay
described above.
Acknowledgements
This study was funded by the UK Biotechnology and
Biological Sciences Research Council. N.S. Scrutton is
a BBSRC Professorial Research Fellow. We thank
K. Marshall for assistance with early parts of the clon-
ing work reported in the article.
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Supporting information
The following supplementary material is available:
Fig. S1. Fluorescence titration of the FMN domain
with the hMS AD.
Fig. S2. Comparison of the electrostatic potentials of
the surface of the CPR FMN domain and of a model
of the FMN domain of human MSR.

Fig. S3. Electrostatic potentials of the surface of the
hMS AD.
Table S1. Sequences of oligonucleotides used for clon-
ing hMS.
Table S2. Sequences of oligonucleotides used in muta-
genesis of cDNA for hMS.
Doc. S1. Additional methods.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supplementary materi-
als supplied by the authors. Any queries (other than
missing material) should be directed to the correspond-
ing author for the article.
K. R. Wolthers and N. S. Scrutton Formation of holo-methionine synthase
FEBS Journal 276 (2009) 1942–1951 ª 2009 The Authors Journal compilation ª 2009 FEBS 1951