Supplementary Information
1. Supplementary Materials and Methods
Cloning of hMS cDNA into Pichia pastoris expression vector. Human cDNA was
prepared from total RNA extracted from human white blood cells using PAXgene Blood
RNA and the Omniscript RT Kits (Qiagen). The cDNA for hMS was amplified in three
segments using the polymerase chain reaction (PCR) and oligonucleotides listed in Table
S1. Primers AsefII and NdeR were used to amplify the first 1126 base pairs (bp) of the
gene (referred to as the AN fragment). Oligonucleotides NdeF and MluR were used to
amplify the middle portion of the gene (1653 bp; termed the NM fragment) and
oligonucleotides MluF and BclI were used to amplify the remaining 1184 bp of the gene
(denoted the MB fragment). The AN fragment was cloned into the NcoI and NdeI site of
pET15d, generating pETAN. The NM and NB fragments were cloned into pGEMT
(Promega, Southampton, UK), to generate pGEMNM and pGEMNB, respectively. The
pGEMNB construct was digested with NdeI and BclI, and the NB fragment was isolated
and sub-cloned into pGEMNM, placing the 3′ end (NB fragment) of the hMS cDNA
downstream of, and in frame with, the middle portion of the gene (i.e. the NM fragment).
The plasmid construct, in which the two fragments (NM and NB) are fused together is
named pGEMMB. Sequencing of pETAN and pGEMMB revealed several missense
mutations according to the published sequence (Genbank accession number: U71285)
which translated into the following amino acid changes: V77A, I273M, I824T, D919G,
D943N, D963E, and K1071N. Oligonucleotides (sequences are listed in Table S2) were
designed to revert these mutations to wild-type sequence using the QuikChange
(Stratagene) protocol.
S1
The AN fragment was subsequently sub-cloned into pGEX4T1. This was
achieved by PCR amplification of this section of cDNA using the oligonucleotides
MSEcoRI and MSXhoR as primers and pETAN as a DNA template. The PCR fragment
was digested with EcoRI and XhoI and ligated into the pGEX4T1 vector, previously
restricted with the same enzymes. The resulting vector was designated pGEXN-term. The
vector pGEMMB was then digested with NotI and EcoRI, and the 2.8 kb fragment
corresponding to the 3’ end of the MS cDNA was isolated by gel electrophoresis,
digested with NotI and NdeI and ligated into pGEXN-term vector also digested with NotI
and NdeI, generating the vector pGEXMS2.
For expression in Pichia pastoris, the hMS cDNA was inserted into pPICZC. To
facilitate this, pGEXMS2 and pPICZC were digested with EcoRI and NotI, and the 3.7 kb
hMS cDNA fragment was ligated with the digested pPICZC to produce pPICZMS. Sitedirected mutagenesis was used to convert the initiation start site to the yeast consensus
sequence, A/YAA/TAATGTCT using the oligonucleotides FMSPCON and RMSPCON
(sequences listed in Table S1 of Supplementary Material). This change did not alter the
hMS codon sequence. To express hMS as a fusion protein with a C-terminal c-myc
epitope tag and (His)6tag, the translational stop codon was converted to a serine residue
by site-directed mutagenesis using oligonucleotides, FMSSTOP and RMSSTOP
(sequences listed in Table S1 of Supplementary Material). The gene encoding hMS
contained in the final construct pPICZMS was sequenced to ensure that PCR induced
errors had not occurred.
S2
Cloning of human MS. Human MS has been cloned previously by three groups [1-4],
and the reported translated sequences are identical (accession numbers U71285, U75743,
and U73338) with one exception. A point mutation in the sequence reported by Li et al
(2) results in conversion of a cysteine residue at position 255 to tyrosine. Sequencing of
our cDNA for hMS revealed several point mutations, which resulted in 7 amino acid
changes, V77A, I273M, I824T, D919G, D943N, D963E, and K1071N. The D919G
exchange, resulting from the A2756G transition, is a polymorphism with an allele
frequency of approximately 15% [1]. It is unclear, if the remaining six mutations were
introduced by PCR amplification or if they originated from the DNA, but it is likely the
latter case as a proofreading DNA polymerase was used (PfuTurbo) and the mutations
were found in several clones. Polymorphisms in genes associated with folate and
homocysteine metabolism, (e.g. methylenetetrahydrofolate reductase,C677T; MSR,
A66G), are linked to an increase risk of certain cancers, cardiovascular disease and
formation of neural tube defects[5, 6]. However, there is no strong evidence to suggest
that the hMS D919G polymorphism affects human health. Several of the identified
sequence differences in our hMS cDNA are relatively conservative (i.e. V77A, D963E,
and K1071N) and therefore may not affect hMS function. That said, we have shown that
mutation of Lys1071 which is located in the activation domain, weakens interaction with
MSR by disrupting electrostatic interaction at the binding interface [7, 8]. To remove any
doubt of these sequence alterations affecting hMS function, they were converted to the
wild-type sequence recorded in Genebank (Accession no. U71285) using standard
mutagenesis procedures.
S3
Table S1. Sequence of oligonucleotides used for cloning hMS.
Primer
Sequence
nt
NdeR*
GGAGACTCATTAATATGTCACCCGCGCTCC
287-302
AseF2
GTTGGTGTACGGTCCAATCCTGAAGG
1381-1405
NdeF
GTTCCACCTGCCACTGCTTTTGAAG
1319-1343
MluR*
GGGAACACACCACCACACTCTTGG
2946-2970
MluF
GTGCACCTGTAATCCATGTCCTGGACG
2915-2942
Bcl1R*
AAAAAATGATCAGTTAGTCTGTATCATATCCCA
4084-4058
AAATGGG
FMSEcoR1
TAGAATTCATGTCACCCGCGCTCCAAGACCTGTCG
RMSXho1*
ATCCTCGAGCATATGTCCTTCAAAAGC
FMSSTOP
GGATATGATACAGACTCACTGATCATTTTTTCATCA
CTAGTGCG GCCGC
RMSSTOP*
GCGGCCGCACTAGTGATGAAAAAATGATCAGTGAG
TCTGTA TCATATCC
FMSPCONC
TAATTATTCGAAACGAGGAAATAATGTCTCCCGCGC
TCCAAGACC
RMSPCON*
GGTCTTGGAGCGCGGGAGACATTATTTCCTCGTTTC
GAATAATTGA
Changes in sequence introduced using primers are shown in bold face type. The numbering
corresponds to the Genebank sequence of MTR, NM000254. The translation start site begins at
base pair 287 in the Genebank sequence. Oligonucleotides with asterisks denote sequences that
are complementary to the noncoding strand, while those without asterisks correspond to the
nucleotide sequence of the noncoding strand of the MTR gene.
S4
Table S2. Sequence of oligonucleotides used in mutagenesis.
Name
Sequence
Mutation
FMSVA
GTATAACTCAGCCTGACGTCATTTACCAAATCCATAAGG
A77V
RMSVA CCTTATGGATTTGGTAAATGACGTCAGGCTGAGTTATAC
A77V
FMSIM
GAAATGAGACCTTTTATTGAAATAATTGGAAAATGTACAACAGC
M273I
RMSIM
GCTGTTGTACATTTTCCAATTATTTCAATAAAAGGTCTCATTTC
M273I
FMSPA
GAGAGCGCTGTAATGTTGCAGGATCAAGGAAGTTTGC
A381P
RMSPA
GCAAACTTCCTTGATCCTGCAACATTACAGCGCTCTC
A381P
FMSDG
GAAGATATTAGACAGGACCATTATGAGTCTCTCAAGGAG
G919D
RMSDG CTCCTTGAGAGACTCATAATGGTCCTGTCTAATATCTTC
G919D
FMSDN
GAAAAAGTGGTTTCCAAATGGATTGGCTGTCTGAACCTCACCC
D943N
RMSDN GGGTGAGGTTCAGACAGCCAATCCATTTGGAAACCACTTTTTC
D943N
FMSDE
GGACCCAGGTCTTTGAAGACTATGACCTGCAGAAGC
E963D
RMSDE
GCTTCTGCAGGTCATAGTCTTCAAAGACCTGGGTCC
E963D
FMSKN
GGCAACAGGCTGAGAAGGACTCTGCCAGCACGG
N1071K
RMSKN CCGTGCTGGCAGAGTCCTTCTCAGCCTGTTGCC
S5
N1071K
 Fluorescence (A.U.)
2. Supplementary data and analysis
8
7
6
5
4
3
2
1
0
0
2
4 6 8 10 12 14 16
[Act domain] M
Figure S1. Fluorescence titration of the FMN-domain with the hMS activation
domain. The FMN-domain (0.25 μM) was titrated with activation domain under the
conditions described in Wolthers and Scrutton [8]. The change in flavin fluorescence
intensity at 529 nm was plotted versus the concentration of the activation domain. A best
fit of the data to a hyperbolic equation previously described [8], gives an apparent
dissociation constant of 1.5 ± 0.1 µM.
S6
CPR FMN domain
Model of the MSR FMN-domain
Figure 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. Using SWISSMODEL [9], a model of the MSR FMN-domain was generated based on the structure of
human CPR-FMN-domain (PDB code: 1b1c). The electrostatic potential surface of CPRFMN domain and the MSR FMN-domain has been calculated using the CCP4 molecular
graphics program. The FMN-cofactor is shown as ball and stick in both images.
Negatively, neutral and positively charged residues of both proteins are coloured in red,
white and blue.
S7
Figure S3. The electrostatic potentials of the surface of the hMS activation domain.
The electrostatic potential surface of hMS activation domain (PDB code: 202K) has been
calculated using the CCP4 molecular graphics program. The substrate, S-adenosylmethionine is shown as ball and stick and negatively, neutral and positively charged
residues on the protein are coloured in red, white and blue.
S8
References
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2
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3
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7
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