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

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

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

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

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-

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

) 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-

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-

) 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-

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

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

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

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.

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]

)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

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)

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

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

)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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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.
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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