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
Trang 1specific 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 (H4-folate) from the
demethy-lation of methyltetrahydrofolate (CH3-H4-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 CH3-H4-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: nigel.scrutton@manchester.ac.uk
(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 (kcat⁄ Km of 1.5· 105m)1Æs)1) and MSR had the lowest Km (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 (Kact 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; FMNhq,FMN hydroquinone; FMNsq,FMN semiquinone; FNR, FAD-dependent ferredoxin–NADP + reductase; H4-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.
Trang 2cob(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 coliMetH, the N-terminal region of hMS comprises
two closely packed (ba)8 barrels that bind
homocyste-ine and CH3-H4-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 Co Methionine
Methylcob(III)alamin Cob(I)alamin
Primary turnover cycle
H CH3 H4folate AdoHyc
Cob(I)alamin Cob(II)alamin
H4folate
Co
NADPH
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
CH3-H4-folate, generating H4-folate and the methylcob(III)alamin form of MS During primary turnover, the homocysteine-binding domain (dot-ted barrel) and the CH3-H4-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].
Trang 3of 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 14CH3 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)1Æmg)1) Yield (%)
Purification n-fold
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.
Trang 4nine from14CH3-H4-folate The rate of14CH3
incorpo-ration was found to saturate with respect to MSR
concentration (Fig 3A) The parameter Kact 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 (kcat) was 1.5 lmolÆmin)1Æmg)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 Km 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 Kd of 37 lm for the MSR–NADP+complex [17], and, by stopped-flow experiments, an apparent Kd
of 50 lm for NADPH for the MSR–NADPH com-plex [18] These values are in reasonable agreement with the apparent Km 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 (FMNsq) or FMN hydroquinone (FMNhq) to the hMS-bound cob(II)alamin [19] Specifically, the mid-point potential values for FMNox⁄ sq and FMNsq⁄ 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 FMNsq or FMNhq] 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 Co3+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 Kactvalue for MSR of 107 ± 1 n M and a Vmax 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 Kmfor NADPH of 23.2 ± 3.4 l M
Trang 5AqCbl 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 FMNsq and FMNhq
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 FMNsq 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 reducreduc-ing hMS and returnreduc-ing 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 (kcat⁄ Km) reveal that CPR has the greatest specificity (10.6· 105m)1Æs)1) for AqCbl, with MSR (4.1· 105m)1Æs)1) and nNOSred (1.5· 105m)1Æs)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 c3+, 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 l M
Ado-Met, 1 m M homocysteine, 250 l M14CH 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.
Without flavoproteina 0.24 ± 0.02
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 reducreduc-ing 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 l M
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 (l M )
k cat ⁄ K m (· 10 5
M )1Æs)1)
Trang 6Holoenzyme 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
37C [14,25] In our studies, hMS activity dropped to
0.01 nmolÆmin)1 in Pichia cell extracts expressing
apo-MS which were incubated at 37C 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 preincubaaddi-tion 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 B12 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 (Kd 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 l M NADPH.
Apo-MS treatment
hMS activity (nmolÆmin)1)
70 min
No Cbl
Without NADPH or MSR 0.01 ± 0.01
AqCbl
Without MSR or NADPH 0.13 ± 0.01
With NADPH and nNOSred 0.18 ± 0.02
MeCbl
Without MSR or NADPH 0.06 ± 0.01
with NADPH and nNOSred 0.19 ± 0.02
Trang 7a 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 FMNsq and FMNhq 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) CH3
-H4-folate and 5-[14C]methyl-H4-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 30C 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 30C 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 CaSO4, 18.2 gÆL)1 K2SO4, 14.9 gÆL)1 MgSO4.7H2O, 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 (PTM1; Invitrogen) The fermentation medium was inocu-lated with 200 mL of starter culture Throughout growth, the temperature was maintained at 29C, 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 PTM1trace salts per litre) was added to the culture at 3.6 mLÆh)1ÆL)1of initial fermentation volume After 1 h, methanol (containing
12 mL of PTM1trace salts per litre) was added to the cul-ture at 1.2 mLÆh)1ÆL)1of initial fermentation volume After
an additional 1 h, the methanol flow rate was increased to 2.4 mLÆh)1ÆL)1and the glycerol feed rate was decreased to 2.4 mLÆh)1ÆL)1 Finally, after another 2 h, the methanol flow rate was increased to 3.6 mLÆh)1ÆL)1of initial fermen-tation volume, and the glycerol feed was terminated The cells continued to grow on methanol supplied at 3.6 mLÆ
h)1ÆL)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 4C, unless otherwise
Trang 8stated Cells (103 g wet weight) were suspended in 250 mL of
50 mm potassium phosphate buffer (pH 7.2), containing
1 mm 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 22C 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 CH3-H4-folate is converted to CH+=H4-folate
[30] The reaction mixture contained 0.1 m potassium
phos-phate buffer (pH 7.2), 0.1 m KCl, 250 l m CH3-H4-folate,
1 mm 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 37C of the
enzyme with all other components Following 10 min of
incubation at 37C, 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 90C for 10 min The
acidification of the reaction mixture quantitatively converts
CH3-H4-folate to CH+=H4-folate, which absorbs strongly
at 350 nm (De = 26 500 m)1cm)1)
Radioactive hMS activity assay
A radioactive hMS assay that monitors the transfer of the
[14C]methyl group from CH3-H4-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 lm14CH3-H4 -folate (1200 d.p.m per nmol), in a total volume of 250 lL All the components were mixed and incubated at 37C for
3 min The reaction was initiated by the addition of 1 mm homocysteine, and then further incubated at 37C for
10 min The reaction was quenched at 90C 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· 1 mL of water The combined radioactivity in the flowthrough and wash fractions was quantitated by scintillation counting For MSR-catalysed assays (used for measuring the Km of NADPH and the Kact 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 (O2>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 O2, 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 lm 14CH3-H4 -folate (1200 d.p.m per nmol), and hMS, in a total volume
of 250 lL The reaction was incubated for 10 min at 37C, 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)1Æcm)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 usfollow-ing a difference extinction coefficient of 5.57· 10)3m)1Æcm)1 [31] on a Cary50 spectrophotometer Reactions were performed in
Trang 950 mm potassium phosphate buffer (pH 7.2), 85 lm
NADPH and variable concentrations of AqCbl, at 37C,
in a total volume of 1 mL The reaction was initiated by
adding 5–20· 1012mol 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)1Æcm)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 37C 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
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