Microbiology (2016), 162, 145–155
DOI 10.1099/mic.0.000209
Physiological role of FolD
(methylenetetrahydrofolate dehydrogenase), FchA
(methenyltetrahydrofolate cyclohydrolase) and
Fhs (formyltetrahydrofolate synthetase) from
Clostridium perfringens in a heterologous model
of Escherichia coli
Srinivas Aluri,1 Shivjee Sah,1 Sandeep Miryala1 and Umesh Varshney1,2
Correspondence
1
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
Umesh Varshney
[email protected]
2
Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India
Received 24 September 2015
Revised
2 November 2015
Accepted 2 November 2015
Most organisms possess bifunctional FolD [5,10-methylenetetrahydrofolate (5,10-CH2-THF)
dehydrogenase-cyclohydrolase] to generate NADPH and 10-formyltetrahdrofolate (10-CHO-THF)
required in various metabolic steps. In addition, some organisms including Clostridium perfringens
possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF.
Here, we show that unlike the bifunctional FolD of Escherichia coli (Eco FolD), and contrary to its
annotated bifunctional nature, C. perfringens FolD (Cpe FolD) is a monofunctional 5,10-CH2-THF
dehydrogenase. The dehydrogenase activity of Cpe FolD is about five times more efficient than
that of Eco FolD. The 5,10-methenyltetrahydrofolate (5,10-CH+-THF) cyclohydrolase activity in
C. perfringens is provided by another protein, FchA (5,10-CH+-THF cyclohydrolase), whose
cyclohydrolase activity is ,10 times more efficient than that of Eco FolD. Kinetic parameters for
Cpe Fhs were also determined for utilization of all of its substrates. Both Cpe FolD and Cpe FchA
are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of
Cpe FolD and Cpe FchA is also necessary to rescue an E. coli folD deletion strain (harbouring
Cpe Fhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to
trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD,
FchA and Fhs) is required to maintain folate homeostasis in the cell.
INTRODUCTION
The one-carbon metabolic pathway (Fig. 1) provides cofactors for the synthesis of glycine, methionine, purines and
thymidylate, and also the formylation of initiator tRNA
(tRNAfMet) in bacteria and eukaryotic organelles. FolD
[methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] is a bifunctional protein in most
organisms (D’Ari & Rabinowitz, 1991; de Mata & Rabinowitz, 1980; Ljungdahl et al., 1980; Murta et al., 2009;
Schmidt et al., 2000). The dehydrogenase activity of FolD
catalyses NADP+-dependent oxidation of 5,10-CH2-THF
to 5,10-methenyltetrahydrofolate (5,10-CH+-THF). The
Abbreviations: 5,10-CH+-THF, 5,10-methenyltetrahydrofolate; 5,10CH2-THF, methylenetetrahydrofolate; 10-CHO-THF, 10-formyltetrahy
drofolate; THF, tetrahydrofolate; TMP, trimethoprim.
Four supplementary figures are available with the online Supplementary
Material.
000209 G 2016 The Authors
latter is then converted to 10-formyltetrahydrofolate
(10-CHO-THF) by the cyclohydrolase activity of the
enzyme. The monofunctional dehydrogenase activities are
found as FolD in some bacteria (Peptostreptococcus and a
few Clostridium spp.) and yMTD in yeast (Barlowe &
Appling, 1990; Ragsdale & Ljungdahl, 1984; Uyeda & Rabinowitz, 1967; Wohlfarth et al., 1991). When organisms
possess FolD with a monofunctional dehydrogenase
activity, additional protein(s) must provide for the cyclohydrolase activity. In Clostridium formicoaceticum, FchA
serves the function of 5,10-CH+-THF cyclohydrolase
(Clark & Ljungdahl, 1982).
In addition to FolD/FchA, organisms may also possess an
alternate mechanism of 10-CHO-THF synthesis from tetrahydrofolate, formate and ATP by utilizing Fhs (formyltetrahydrofolate synthetase) (Paukert & Rabinowitz, 1980).
However, unlike FolD, Fhs distribution is not ubiquitous
(Sah et al., 2015). Although the requirement for
10-CHO-THF in cells can be met by the dehydrogenase/
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145
S. Aluri and others
cyclohydrolase activities of FolD/FchA, many organisms
retain both the dehydrogenase/cyclohydrolase- and Fhsmediated mechanisms. In Streptococcus mutans, fhs is
essential and its deletion results in purine auxotrophy
(Crowley et al., 1997). It has also been observed that the
presence of Fhs (in addition to FolD) confers a growth
advantage to Escherichia coli under hypoxia (Sah et al.,
2015).
DHF
FolD and MTHFD (the eukaryotic counterpart) play an
important role in maintaining the NADP+/NADPH ratio
(Fan et al., 2014; Sah et al., 2015). In E. coli, it has been
reported that a folD deletion strain (harbouring Clostridium perfringens Fhs) was auxotrophic for glycine and
purine/formate (Sah et al., 2015). In addition, the strain
showed hypersensitivity to trimethoprim. However,
which of the activities (dehydrogenase and/or cyclohydrolase) of FolD contributed to the growth defects of the E. coli
strain deleted for FolD has not been investigated. Although
the dehydrogenase function of FolD makes a major contribution to the production and maintenance of NADPH
levels in cells, the role of cyclohydrolase activity in this
regard could also be very important in driving the reaction
in the forward direction (Fig. 1).
THF
E. coli, a commonly used model, possesses folD (encoding a
bifunctional FolD), but lacks fhs. However, C. perfringens
possesses folD, fchA and fhs genes. Although preliminary
studies on bifunctional FolD from Clostridium thermoaceticum (Ljungdahl et al., 1980), monofunctional FolD from
Clostridium cylindrosporum (Uyeda & Rabinowitz, 1967),
FchA from C. formicoaceticum (Clark & Ljungdahl, 1982),
and Fhs from C. cylindrosporum, Clostridium acidi-urici
(Himes & Rabinowitz, 1962) and C. cylindrosporum
(Joyce & Himes, 1966) have been carried out, their detailed
kinetic parameters and physiological roles have not been
investigated.
Fig. 1. Schematic of the one-carbon metabolic pathway. The
pathway includes dihydrofolate reductase (FolA), serine hydroxymethyltransferase (GlyA), 5,10-methylenetetrahydrofolate dehydrogenase-cyclohydrolase (FolD), 5,10-methenyltetrahydrofolate
cyclohydrolase (FchA), formyltetrahydrofolate synthetase (Fhs),
10-formyltetrahydrofolate : L -methionyl-tRNAfMet N-formyltransferase (Fmt), phosphoribosylglycinamide (GAR) formyltransferase
(PurN), 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)
transformylase/IMP cyclohydrolase (PurH), 5,10-methylenetetrahydrofolate reductase (MetF), thymidylate synthase (ThyA), and
methionine synthase (MetE/MetH). DHF, dihydrofolate.
In this study, we carried out in vitro and in vivo characterization of FolD, FchA and Fhs from C. perfringens
(Cpe FolD). We show that Cpe FolD is a monofunctional
dehydrogenase. Both Cpe FolD and Cpe FchA are required
to support folD deletion in E. coli.
ThyA
dTMP
NADPH
dUMP
5-CH3-THF
FolA
NADP+
tE ,
Me
etH
M
Methionine
NAD+
ine
ste
cy
mo
MetF
NADH
Ho
5,10-CH2-THF
GlyA
Serine
NADP+
Glycine
FolD
Met-tRNAfMet
NADPH
5,10-CH+-THF
fMet-tRNAfMet Fmt
H2O
Formate+ATP
FolD/FchA
H+
ADP+Pi
10-CHO-THF
Fhs
GAR
PurN
FGAR
AICAR
PurH
FAICAR
F
medium. E. coli cultures were grown at 37 uC with constant shaking at
200 r.p.m. The growth curves were prepared as semi-log plots with
GraphPad Prism using a log2 scale for the y-axis.
Genetic manipulations. RbCl- or CaCl2-based methods were used
to transform E. coli (Sambrook & Russell, 2001). P1 phage-mediated
transductions were used to transfer genetic material between strains
(Miller, 1972).
METHODS
Materials. Media components (Difco) were supplied by BD Bios-
ciences. The enzymes used for various DNA manipulations were
obtained from Finnzymes, New England Biolabs or Roche. Chemicals
of molecular or analytical grade were obtained from Sigma, GE
Healthcare or Qualigens. (6R,S)-Tetrahydrofolate (THF) and DNA
oligomers were from Sigma-Aldrich. (6R,S)-5,10-CH2-THF (calcium
salt) and (6R,S)-5,10-CH+-THF chloride were from Schircks
Laboratories.
Growth and culture conditions. Bacterial strains and plasmids used
are listed in Table 1. All strains were grown in Luria–Bertani broth
(LB), LB-agar (1.8 % agar) or M9 minimal media (with 0.4 % glucose
as carbon source) containing 1 mg thiamine ml21 (Sambrook &
Russell, 2001). When required, ampicillin (100 mg ml21), kanamycin
(25 mg ml21) or tetracycline (7.5 mg ml21) were added to the
146
Generation of plasmid constructs. All plasmid constructs were
made using standard recombinant DNA methods (Sambrook &
Russell, 2001). Pfu or Taq DNA polymerases were used for PCR.
The ORF of fhs was subcloned from pCpe Fhs (Table 1) into
pET14b between the Nde I and Eco RI sites. Cpe-folD was amplified
from C. perfringens ATCC 13124 genomic DNA (available in the
laboratory) with Pfu DNA polymerase using Cpe folD fp
(59-CAGGCCATGGATAAAATTTTAAG-39) and Cpe folD rp
(59-ATAATGAATTCAGTTTCATTCTAATC-39) primers. PCR involved 30 cycles of incubations at 94 uC for 1 min, 42 uC for 30 s
and 70 uC for 2 min. The amplicon was treated with Nco I and Eco RI,
and cloned between the Nco I and Eco RI sites of pBAD HisB vector
to generate pBAD-Cpe FolD. For generating pQE-Cpe FolD, Cpe-folD
was amplified using Cpe folD fp and Cpe folD Bgl II rp
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Microbiology 162
Characterization of C. perfringens FolD, FchA and Fhs
Table 1. E. coli strains and plasmids
Strain/plasmid
Strain
TG1
TG1DfolD/pCpe Fhs
TG1DfolD-fhs
KL16
KL16DfolD/pCpe Fhs
JW2535-1 (DglyA)
KL16DglyA
BL21(DE3)pLysS Rosetta
Plasmid
pET14b
pET14b-Cpe Fhs
pPROEXHTb
pPROEXHTb-Cpe FchA
pTrc99C
pCpe FchA
pQE60
pQEEco FolD
pQECpe FolD
pCpe FchA-FolD
pBAD HisB
pBADCpe FolD
pACDH
pCpe Fhs
Genotype/details
2
K-12 supE thi-1 D(lac-proAB) D(mcrB–hsdSM)5, (r2
K mK ), F9 [traD36
+
q
proAB lacI lacZDM15 ]
E. coli TG1 deleted for folD ( folD : : kan) in the presence of pCpe Fhs
support plasmid
Derivative of TG1DfolD supported by single-copy insertion of Cpe-fhs at
chb locus (Dchb : : fhs-kan R)
l 2, e14 –, relA1, spoT1, thiE1
E. coli KL16 deleted for folD ( folD : : kan) in the presence of pCpe Fhs
support plasmid
F2, D(araD–araB)567, DlacZ4787( : : rrnB-3), l, DglyA725 : : kan, rph-1,
D(rhaD–rhaB) 568, hsdR514
DglyA725 : : kan allele from JW2535-1 was moved to KL16 using P1
phage-mediated transduction
lon-11, D(ompT–nfrA)885, D(galM–ybhJ)884, lDE3 [lacI, lacUV5-T7 gene
1, ind1, sam7, nin5 ], D46, [mal +]K-12(l S), hsdS10 harbouring
pLysSRARE for regulated T7 RNA polymerase and improved reading
of rare codons
T7 RNA polymerase-based expression vector that provides a His-tag for
the cloned ORF for affinity purification using Ni-NTA
Cpe Fhs ORF was subcloned from pCpe Fhs into pET14b between Nde I
and Eco RI sites
E. coli RNA polymerase-based expression vector useful to add a
N-terminal His-tag to the cloned ORF
Cpe FchA between Nco I and Xba I sites in pPROEXHTb vector
E. coli RNA polymerase-based medium-copy expression vector
(ColE1 ori) with cloning sites
Cpe FchA ORF between Nco I and Xba I sites in pTrc99c
Expression vector harbouring T5 promoter for E. coli RNA polymerase
Renamed from p-folD; Eco FolD ORF between Nco I and Bgl II sites of
pQE60
Cpe FolD ORF between Nco I and Bgl II sites of pQE60
Cpe FolD (together with a promoter and terminator elements of pQE60)
between Pst I and Xba I sites of pCpe FchA
Medium-copy expression vector (ColE1 ori) harbouring araBAD
promoter (pBAD) for tightly regulated expression of the cloned ORFs
C. perfringens folD ORF between Nco I and Eco RI sites of pBAD HisB
Plasmid with ACYC ori of replication, compatible with ColE1 ori
plasmids (TetR)
Renamed from p-fhs; Cpe Fhs ORF between Nco I and Eco RI sites of
pACDH
(59-TTTCAGATCTATCTAACTCCTTAC-39), digested with Nco I and
Bgl II, cloned between the same sites of pQE60 and verified by
sequencing.
fchA ORF was PCR amplified from C. perfringens genomic DNA with
Taq DNA polymerase using Cpe fchA fp (59-ACACCATGGAAAACGAAAAG-39) and Cpe fchA rp (59-TATTTATCTAGAAATTTTT
ATTCTATTTTTAC-39). PCR involved 30 cycles of initial denaturation at 94 uC for 1 min, annealing at 44 uC for 30 s and extension at 70 uC for 1 min. The amplicon was digested with Nco I and
Xba I, and ligated to similarly digested pTrc99C to generate
pCpe FchA. To generate pPROEXHTb-Cpe FchA, pCpe FchA was
digested with Nco I and Xba I, and Cpe-fchA was subcloned into a
similarly digested pPROEXHTb.
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Reference/source
Sambrook et al. (1989)
Sah et al. (2015)
Sah et al. (2015)
Low (1968)
This study
Baba et al. (2006)
This study
Novagen
Novagen
This study
Invitrogen
This study
GE Life Sciences
This study
Qiagen
Sah et al. (2015)
This study
This study
Invitrogen
This study
Rao & Varshney (2002)
Sah et al. (2015)
To generate a construct with both Cpe-fchA and Cpe-folD, Cpe-folD
was amplified from pQE-Cpe FolD along with promoter and terminator elements using Pfu DNA polymerase, pQE Sc FP (59-ATTAACCTGCAGAAATAGGCGTAT-39) and pQE Sc Xba I RP
(59-CAGTCAGTTTCTAGATGTACCTATAA-39). PCR involved 30
cycles of denaturation at 94 uC for 1 min, annealing at 50 uC for 30 s
and extension at 70 uC for 3 min. The amplicon was digested with
Pst I and Xba I, and ligated in similarly digested pCpe FchA to generate
pCpe FchA-FolD.
Purification of EcoFolD, CpeFolD, CpeFchA and CpeFhs. Eco -
FolD was purified using the pQE-Eco FolD expression construct from
E. coli TG1DfolD/pCpe Fhs (Sah & Varshney, 2015). To purify
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S. Aluri and others
Cpe FolD and Cpe FchA, expression constructs pQE-Cpe FolD and
pPROEXHTb-Cpe FchA, respectively, were introduced into E. coli
TG1DfolD-fhs (Table 1), and to purify Cpe Fhs, pET14b-Cpe Fhs was
introduced into E. coli BL21(DE3)pLysS Rosetta. Transformants
harbouring expression plasmids were inoculated in 3 l of LB and
grown to OD600 0.6, induced with 0.5 mM IPTG and grown further
for 3 h. Cells were pelleted at 5000 g for 5 min at 4 uC. Unless stated
otherwise, all the following steps were carried out at 4 uC. The cell
pellet was suspended in 10 ml of buffer A [50 mM Tris/HCl
(pH 7.5), 0.3 M KCl, 10 mM b-mercaptoethanol and 10 % glycerol]
and sonicated for 3 min whilst maintaining the temperature at
v10 uC. The resulting extract was spun at 10 000 g for 20 min to
remove cell debris. Approximately 10 ml of the supernatant was
loaded onto a HisTrap column (GE Healthcare, 5 ml) using a
peristaltic pump at a flow rate of 1 ml min21. After an extended
wash with buffer A, a linear gradient of 0–1 M imidazole was
developed over 6 column volumes in 30 min. The fractions containing the required protein were pooled and concentrated using
Centricon YM-10 centrifugal filter, and then stored in the same
buffer containing 50 % glycerol at 220 uC.
RESULTS
Purification of CpeFolD, CpeFchA, CpeFhs and
EcoFolD
Eco FolD, Cpe FolD and Cpe FchA were purified to near
homogeneity (Fig. S1, available in the online Supplementary Material) using DfolD/pCpe Fhs or DfolD-fhs derivatives of E. coli TG1 to ensure no carryover of the host
FolD in the preparations. Cpe Fhs was also purified to
near homogeneity (Fig. S1) using E. coli BL21 host,
which naturally lacks the fhs gene. The recombinant proteins possessed a His6-tag to facilitate purification by
Ni-NTA chromatography.
Cpe FolD possesses monofunctional activity of
5,10-CH2-THF dehydrogenase
Enzyme assays and kinetics. Dehydrogenase activities of Cpe -
In all databases, C. perfringens FolD (Cpe FolD) has been
annotated as a bifunctional 5,10-CH2-THF dehydrogenase-cyclohydrolase. However, the sequence alignment of
Cpe FolD with Eco FolD (Fig. 2) revealed that it lacked
some key residues. Amongst these, K54 and Q98 (E. coli
numbering) important for cyclohydrolase activity were
substituted with Q and L, respectively, in Cpe FolD. Furthermore, G122 in the KDVDG motif of Eco FolD was represented by C in Cpe FolD. These observations raise a
question about the biochemical activities of the clostridial
FolD proteins.
Cyclohydrolase activities of Cpe FchA and Eco FolD were monitored
for 30 s for a decrease in the A355 of (6R,S)-5,10-CH+-THF (5–
113 mM), and the amount of substrate decrease was estimated using
the extinction coefficient (e355524 900 M21 cm21 at neutral pH). Fhs
(0.5 mg) assays contained 50 mM sodium formate, 2.5 mM ATP,
0.5 mM (6R,S)-THF, 20 mM b-mercaptoethanol, 50 mM KCl,
40 mM MgCl2 and 50 mM Tris/HCl (pH 8.2) together with the
purified Cpe Fhs. Perchloric acid was added to a final concentration of
0.5 % to stop the reaction and to convert 10-CHO-THF to 5,10CH+-THF which was measured using its extinction coefficient
(e350524 900 M21 cm21 at acidic pH). The Km and Vmax values for
(6R,S)-THF were determined by using a fixed concentration of ATP
(2.5 mM) and formate (50 mM). The concentration of (6R,S)-THF
was varied from 100 to 2500 mM. The kinetic constants for formate
were determined at a fixed concentration of (6R,S)-THF (1.2 mM)
and ATP (2.5 mM), and varying the formate concentration in the
range 1–100 mM. The kinetic constants for ATP were determined by
keeping a fixed concentration of (6R,S)-THF (1.2 mM) and formate
(50 mM), and varying the ATP concentration in the range 25–
600 mM.
Recently, we showed that deletion of folD in E. coli (with fhs
support, DfolD/pCpe Fhs) resulted in auxotrophy for formate and glycine, which could be rescued upon Eco FolD
expression (Sah et al., 2015). Thus, to test for its in vivo
function, Cpe FolD was expressed in E. coli DfolD/pCpe Fhs
from pBAD-Cpe FolD. However, it failed to rescue the
strain for its requirements for formate and glycine (Fig.
S2). Consistent with the observations made from the CLUSTAL W alignment (Fig. 2), this finding suggested that Cpe FolD lacked either or both of the dehydrogenase and
cyclohydrolase activities. Biochemical assays showed that
Cpe FolD possessed dehydrogenase but not the cyclohydrolase activities (Fig. 3a, b). To investigate further, we determined the kinetic parameters of Cpe FolD (Fig. 4a, b),
which revealed that it utilized 5,10-CH2-THF and
NADP+ with a Km of 228 and 85 mM, respectively, and a
Vmax of 56 and 52 mmol min21 mg21, respectively, for
the two substrates.
FolD (0.04 mg), Eco FolD (0.4 mg) and Cpe FchA (60 mg) and
cyclohydrolase activities of Cpe FchA (0.1 mg), Cpe FolD (0.65 mg),
Eco FolD (1 mg) were assayed in 0.1 M potassium maleate (pH 7.6)
(Sah & Varshney, 2015). Dehydrogenase activity of FolD was
monitored spectrophotometrically by NADP+-dependent oxidation of (6R,S)-5,10-CH2-THF. The Km and Vmax values for
(6R,S)-5,10-CH2-THF were determined by using a fixed concentration of NADP+(2 mM). The concentration of (6R,S)-5,10-CH2THF was varied from 10 to 2000 mM. The kinetic constants for
NADP+ were determined at a fixed concentration of (6R,S)-5,10CH2-THF (2 mM) and varying the NADP+ concentration in the
range of 10–2000 mM.
Graphs for the determination of kinetic constants were prepared by
using GraphPad Prism software.
Photorepair assay. Saturated cultures of E. coli strains were seri22
27
ally diluted (10 to 10 ) in LB with appropriate antibiotic(s).
Aliquots (200 ml) of each dilution were serially pipetted into a 96well ELISA plate and spotted using a 48-pronged spotter (Sigma)
onto LB agar plates containing the required antibiotics. After
spotting, one plate was not exposed and the other plates were
exposed to UV light (UV-C) at different intensities (as indicated).
After exposure, plates were incubated in white light at 37 uC for
15 h.
148
Cpe FchA carries out the cyclohydrolase activity
In the C. perfringens genome database, FchA (Fig. S3) has
been annotated as 5,10-CH+-THF cyclohydrolase.
As shown in Fig. 3(a, b), Cpe FchA showed cyclohydrolase
activity but not dehydrogenase activity. Determination of
the kinetic parameters (Km and Vmax) of the enzyme
revealed that it possessed a Km of 157 mM and a Vmax of
573 mmol min21 mg21 for 5,10-CH+-THF (Fig. 4c).
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Characterization of C. perfringens FolD, FchA and Fhs
50
Cth
Eco
Cpe
54
1
1
1
59
60
58
98
Cth 60
Eco 61
Cpe 59
116
117
115
122
121
Cth 117
Eco 118
Cpe 116
172
173
171
Fig. 2. Multiple sequence alignment of FolD. Amino acid sequences of FolD from different organisms were aligned using
CLUSTAL W and the BOXSHADE 3.21 server. Identical residues are shown in black; similar residues are shown in light grey
boxes. Residues involved in substrate binding/catalysis are shown in boxes. Important residues missing/substituted are
shown by filled triangles. Positions of K54, Q98, D121 and G122 have been marked according to the E. coli numbering
scheme. Cth, C. thermoaceticum (Morella thermoacetica); Eco, E. coli; Cpe, C. perfringens.
Thus, Cpe FchA might supplement for the missing cyclohydrolase activity of Cpe FolD.
for (6R,S)-THF, formate and ATP were 52, 36 and
37 mmol min21 mg21, respectively (Fig. 5).
Biochemical characterization of Cpe Fhs
Simultaneous presence of Cpe FolD and
Cpe FchA rescues the DfolD strain of E. coli for
its requirements for formate and glycine
In addition to FolD/FchA, C. perfringens possesses Fhs,
which also contributes to 10-CHO-THF synthesis.
In bacteria growing under anaerobic conditions, Fhs contributes toward much of the 10-CHO-THF synthesis.
As Fhs plays a key role in one-carbon metabolism of
anaerobic bacteria, it was important to determine its Km
and Vmax values. Kinetic constants were determined for
substrates and cofactors of the Cpe Fhs reaction. Km
values for (6R,S)-THF, formate and ATP were found to
be 330 mM, 3 mM and 91 mM, respectively. Vmax values
(b)
80
60
40
20
0
CpeFoID
EcoFoID
CpeFchA
Activity [µmol 10-CHO-THF min–1
(mg protein)–1]
Activity [µmol 5,10-CH+-THF min–1
(mg protein)–1]
(a)
Biochemical assays (Figs 3 and 4) showed that Cpe FolD
and Cpe FchA possess dehydrogenase and cyclohydrolase
activities, respectively. As the presence of Cpe FolD alone
(Fig. S2) did not rescue E. coli for its deficiency of FolD,
it was of interest to test whether the simultaneous presence
of Cpe FolD and Cpe FchA would rescue the DfolD/
pCpe Fhs strain for its requirements for formate and glycine. For this purpose, Cpe-fchA and Cpe-folD were
cloned into a medium-copy plasmid, pTrc99C, to generate
250
200
150
100
50
0
CpeFoID
EcoFoID
CpeFchA
Fig. 3. Cpe FolD and Cpe FchA are monofunctional enzymes. (a) Dehydrogenase and (b) cyclohydrolase activities were
measured for Eco FolD, Cpe FolD and Cpe FchA (see Methods). Cpe FolD has only dehydrogenase activity, whereas
Cpe FchA has only cyclohydrolase activity. Eco FolD is a bifunctional enzyme for dehydrogenase and cyclohydrolase activities.
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S. Aluri and others
40
Vmax: 56±2 µmol min–1 mg–1
20
Km: 228±24 µM
0
0
500 1000 1500 2000 2500
(c)
60
40
Vmax: 52±1 µmol min–1 mg–1
20
Km: 85±5 µM
0
0
500 1000 1500 2000 2500
(6R,S)-5,10-CH2-THF (μM)
Activity [µmol 10-CHO-THF
min–1 (mg protein)–1]
60
Activity [µmol 5,10-CH+-THF
min–1 (mg protein)–1]
(b)
Activity [µmol 5,10-CH+-THF
min–1 (mg protein)–1]
(a)
300
Vmax: 573±123 µmol min–1 mg–1
Km: 157±50 µM
200
100
0
0
NADP+ (μM)
20
40
60
80
100 120
(6R,S)-5,10-CH+-THF (μM)
Fig. 4. Michaelis–Menten plot for the kinetics of Cpe FolD and Cpe FchA. (a, b) Kinetic constants for Cpe FolD dehydrogenase were obtained by performing a dehydrogenase assay where the concentration of one substrate was varied at a fixed concentration of the other substrate: (a) (6R,S)-5,10-CH2-THF and, (b) NADP+. (c) Kinetic constants for Cpe FchA were
obtained by performing a cyclohydrolase assay where the concentration of the substrate (6R,S)-5,10-CH+-THF was varied
at a fixed concentration of the enzyme (Methods).
pCpe FchA-FolD and introduced into the DfolD/pCpe Fhs
strain. As shown in Fig. 6, the simultaneous presence of
Cpe FolD and Cpe FchA supported the growth of the
DfolD/pCpe Fhs strain in M9 minimal medium, and rescued it for its requirements for formate and glycine. Induction of proteins with 0.1 mM IPTG further accelerated the
growth of the strain.
substitute for the essential function of a bifunctional FolD
(Fig. S4). Growth analyses of the transductants also
showed that Cpe FolD and Cpe FchA indeed supported
the DfolD strain in LB (Fig. 7a). When compared with
E. coli KL16 (WT for folD), pCpe FchA-FolD-supported
strains showed a decrease in growth in LB medium, but
addition of 0.1 mM IPTG restored the growth to almost
the same as that of the WT strain (compare Fig. 7a and
b). A similar phenotype was also observed for the
growth in M9 minimal medium (Fig. 7c), although
pCpe FchA-FolD-supported strains showed a larger
decrease in growth in M9 medium. Whilst addition
0.1 mM IPTG rescued the growth (compare Fig. 7c and
d), it still did not reach the growth seen for the WT
strain. This could be due to the increased requirements
for the metabolites synthesized via the folate pathway.
Cpe FolD and Cpe FchA together substitute for
the function of FolD in E. coli
The result in Fig. 6 suggested that the simultaneous presence of Cpe FolD and Cpe FchA might allow for the deletion of folD (without Cpe Fhs support) from E. coli.
To test this, we used P1 phage-mediated transductions
to replace the genomic copy of folD with a kan marker
( folD : : kan) in E. coli KL16 harbouring pCpe FchAFolD. Deletion of folD could be achieved in the presence
of pCpe FchA-FolD, suggesting that the monofunctional
dehydrogenase and cyclohydrolase activities of Cpe FolD
and Cpe FchA, respectively, when present together could
We have previously shown that folD deletion (in the presence of Cpe Fhs) conferred hypersensitivity to TMP due
40
30
20
Vmax: 52±3 µmol min–1 mg–1
10
Km: 330±67 µM
0
0
1000
2000
(6R,S)-THF (μM)
3000
(c)
40
30
20
Vmax: 36±3 µmol min–1 mg–1
Km: 3±0.3 µM
10
0
0
25
50
75
100
125
Activity [µmol 10-CHO-THF
min–1 (mg protein)–1]
(b)
50
Activity [µmol 5,10-CHO-THF
min–1 (mg protein)–1]
Activity [µmol 10-CHO-THF
min–1 (mg protein)–1]
(a)
Cpe FolD and Cpe FchA rescue the DfolD strains
for TMP hypersensitivity
Formate (mM)
40
30
20
Vmax: 37±2 µmol min–1 mg–1
Km: 91±14 µM
10
0
0
200
400
600
800
ATP (μM)
Fig. 5. Michaelis–Menten plot of the kinetics of Cpe Fhs. Kinetic constants were obtained by performing an Fhs assay where
the concentration of one substrate was varied at a fixed concentration of the other two substrates (Methods): (a) (6R,S)-THF,
(b) formate and (c) ATP.
150
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1
ΔfolD/pCpeFhs/vector
ΔfolD/pCpeFhs/pCpeFoID-FchA
0.5
0.25
0.125
(b)
2
1
ΔfolD/pCpeFhs/vector
ΔfolD/pCpeFhs/pCpeFoID-FchA
0.5
0.25
0.125
(c)
2
Growth (OD600)
2
Growth (OD600)
(a)
Growth (OD600)
Characterization of C. perfringens FolD, FchA and Fhs
1
0
5
10
15
20
25
30
35
0.5
0.25
0.125
0.0625
0.0625
0.0625
ΔfolD/pCpeFhs/vector
ΔfolD/pCpeFhs/pCpeFoID-FchA
0
5
10
15
Time (h)
20
25
30
35
0
5
Time (h)
10
15
20
25
30
35
Time (h)
Fig. 6. Cpe FolD and Cpe FchA rescue the DfolD/pCpe Fhs strain for its requirement for formate and glycine. The E. coli
KL16DfolD/pCpe Fhs strain harbouring vector alone (pTrc99C) or its derivative pCpe FchA-FolD was inoculated in (a) M9
minimal media, M9 minimal media supplemented with (b) IPTG or (c) formate (10 mM) and glycine (0.3 mg ml21) and monitored for their growth.
Cpe Fhs and Cpe FolD-FchA rescue the
photosensitive phenotype
to altered folate metabolism and THF deficiency in the
cell (Sah et al., 2015). TMP, an antifolate, inhibits dihydrofolate reductase (FolA). As shown in Fig. 8,
expression of Cpe FolD and Cpe FchA together also partially rescued the strain for TMP hypersensitivity,
suggesting that the simultaneous presence of both proteins is important in maintaining folate homeostasis
and antifolate resistance.
(b)
2
2
1
1
Growth (OD600)
Growth (OD600)
(a)
5,10-CH+-THF, a cofactor used by photolyase and involved
in the direct repair of pyrimidine dimers, is synthesized
by FolD (from 5,10-CH2-THF or 10-CHO-THF by its
dehydrogenase and reverse cyclohydrolase activities, respectively). Synthesis of 5,10-CH2-THF and 5,10-CH+-THF
0.5
0.25
0.125
KL16 (WT)
0.5
ΔfolD/pCpeFchA-FoID
0.25
Media control
0.125
0.0625
0.0625
0
10
20
30
0
10
Time (h)
(c)
30
(d)
2
2
1
1
Growth (OD600)
Growth (OD600)
20
Time (h)
0.5
0.25
0.125
0.0625
KL16 (WT)
0.5
ΔfolD/pCpeFchA-FoID
0.25
Media control
0.125
0.0625
0
10
20
Time (h)
30
0
10
20
30
Time (h)
Fig. 7. Cpe FolD and Cpe FchA allow folD deletion in E. coli. (a, b) E. coli KL16 and KL16DfolD/pCpe FchA-FolD strains
were inoculated in LB medium and followed for their growth (a). IPTG was added to a final concentration of 0.1 mM (b).
(c, d) KL16 and KL16DfolD/pCpe FchA-FolD strains were inoculated in M9 minimal medium and followed for their growth (c).
IPTG was added to a final concentration of 0.1 mM (d).
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151
1
0.5
0.25
KL16
ΔfolD/pCpeFhs/vector
0.125
ΔfolD/pCpeFhs/pCpeFhs-FoID
(b)
2
KL16
ΔfolD/pCpeFhs/vector
1
ΔfolD/pCpeFhs/pCpeFhs-FoID
0.5
0.25
0.125
0.0625
0.0625
0
10
20
30
(c)
2
KL16
ΔfolD/pCpeFhs/vector
Growth (OD600)
2
Growth (OD600)
(a)
Growth (OD600)
S. Aluri and others
1
ΔfolD/pCpeFhs/pCpeFhs-FoID
0.5
0.25
0.125
0.0625
0
10
Time (h)
20
30
0
Time (h)
10
20
30
Time (h)
Fig. 8. Cpe FolD and Cpe FchA rescue TMP sensitivity of the DfolD/pCpe Fhs strain. (a) The KL16DfolD/pCpe Fhs strains
harbouring various plasmids were inoculated in LB. (b, c) TMP was added at 0.6 (b) and 1.2 (c) mg ml21 and the cultures
were followed for their growth.
is regulated by GlyA. An E. coli DglyA strain is also defective
in 5,10-CH+-THF and UV photosensitive (Fig. 9a, compare row 3 with row 1). FolD can also synthesize 5,10CH+-THF from 10-CHO-THF by the reversible step of
the cyclohydrolase reaction, provided sufficient amounts
of the latter are present. Expression of Fhs could increase
the steady-state levels of 10-CHO-THF and drive the reaction toward the synthesis of 5,10-CH+-THF. Thus,
expression of Fhs in E. coli DglyA could rescue UV photosensitivity of the strain. Indeed, we saw that expression of
(a)
10–2 10–3 10–4 10–5 10–6
10–2 10–3 10–4 10–5 10–6
1
pACDH
2
pCpeFhs
3
ΔglyA/pACDH
ΔglyA/pCpeFhs
4
Unexposed
2 mJ cm–2
(b)
10–1 10–2 10–3 10–4 10–5
10–1 10–2 10–3 10–4 10–5 10–1 10–2 10–3 10–4 10–5
10–1 10–2 10–3 10–4 10–5
1
KL16
2
KL16/pCpeFhs
3
ΔfolD/pCpeFchA-FolD
4
ΔfolD/pCpeFhs/pTrc99C
5
ΔfolD/pCpeFhs/pCpeFchA
6
ΔfolD/pCpeFhs/pCpeFolD
7
ΔfolD/pCpeFhs/pEcoFolD
8
ΔfolD/pCpeFhs/pCpeFchA-FolD
Unexposed
1 mJ cm–2
2.5 mJ cm–2
5 mJ cm–2
Fig. 9. (a) Rescue of UV photosensitivity of the DglyA strain. Saturated cultures of E. coli KL16DglyA harbouring the indicated plasmids were serially diluted (1022 to 1027) and spotted using a 48-pronged spotter (Sigma) onto LB agar plates.
After spotting, one plate was left unexposed and the other plate was exposed at an intensity of 2 mJ cm22. After exposure,
the plates were incubated at 37 8C for 15 h in white light. (b) Cpe-folD and Cpe-folD-fchA rescue UV photosensitivity of the
DfolD/pCpe Fhs strain of E. coli. UV photosensitivity assay using E. coli KL16 or its DfolD derivative harbouring different plasmids (as indicated) was carried out. Saturated cultures were serially diluted (1021 to 1025 ) and spotted using a 48-pronged
spotter (Sigma) onto LB agar plates. After spotting, one plate was not exposed and the other plates were exposed to UV
light (UV-C) at different intensities (as indicated). After exposure, the plates were incubated in the presence of white light at
37 8C for 15 h.
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Microbiology 162
Characterization of C. perfringens FolD, FchA and Fhs
Cpe Fhs led to rescue of UV photosensitivity of the DglyA
strain (Fig. 9a, compare row 4 with row 3), suggesting
that Cpe Fhs synthesizes 10-CHO-THF, which contributes
toward the synthesis 5,10-CH+-THF and rescues UV
photosensitivity of the DglyA strain of E. coli. As controls,
KL16/pACDH and KL16/pCpe Fhs did not show any
growth difference (Fig. 9a, rows 1 and 2).
The DfolD strain (in the presence of Fhs) was photosensitive
(Fig. 9b, compare row 4 in cultures not exposed to UV with
those exposed to UV at 1, 2.5 and 5 mJ cm22). This was
expected due to the deficiency of 5,10-CH+-THF in the
absence of both the dehydrogenase and cyclohydrolase
activities of Eco FolD. To check further for the compatibility
of the clostridial proteins (FolD, FchA and Fhs) in E. coli, we
tested if the clostridial proteins rescued the photorepair
deficiency of the DfolD strain. Whilst the expression of
FchA alone did not rescue the photosensitive phenotype
of the DfolD strain (Fig. 9b, compare row 5 with row 4 at
1 or 2.5 mJ cm22), expression of Cpe FolD alone (Fig. 9b,
compare row 6 with row 4) or Cpe FolD and Cpe FchA
together (Fig. 9b, compare row 8 with row 4) did. In fact,
simultaneous expression of Cpe FolD and Cpe FchA even
in the background lacking Fhs (Fig. 9b, compare row 3
with row 4) rescued the photorepair deficiency. Furthermore, as the simultaneous expression of both the proteins
resulted in a better rescue than Cpe FolD alone did, this indicated that the presence of the Cpe FchA function (together
with Cpe FolD) was essential for the steady-state supply of
folate intermediates. As a control, we observed that the
photosensitive phenotype of the strain could be restored
by expressing Eco FolD (Fig. 9b, compare row 7 with row 4).
DISCUSSION
Folate metabolism is important in all organisms in providing folate cofactors for the synthesis of purines, thymidylate, glycine and methionine, and also for the formylation
of tRNAfMet (in bacteria and eukaryotic organelles).
As purine synthesis is required to occur at a high rate in
all cells, the cofactors involved in the pathway must be
available in abundance. 10-CHO-THF is one of the important folate cofactors required for purine biosynthesis.
Enzymes involved in folate metabolism have been studied
extensively from different genera of bacteria. However, in
bacteria such as the species of genus Clostridium, which
are obligate anaerobes, study of folate metabolism in vivo
has been limited due to the strict anaerobic nature of
their growth. For this reason, many enzymes have been studied in vitro. Amongst the enzymes involved in folate
metabolism, Fhs has been characterized from various
non-pathogenic species of Clostridium (Curthoys &
Rabinowitz, 1972). Likewise, whilst monofunctional 5,
10-CH2-THF dehydrogenase and 5,10-CH+-THF cyclohydrolase were reported from C. cylindrosporum (Uyeda &
Rabinowitz, 1967) and C. formicoaceticum (Clark & Ljungdahl, 1982), a detailed kinetic analysis was not performed.
Here, we investigated the in vitro and in vivo properties of
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FolD, FchA and Fhs from C. perfringens, a pathogen, using
E. coli as a surrogate model. Study of these enzymes is
important as they play a central role in folate homeostasis
and serve as targets for antibacterials. In the National
Center for Biotechnology Information and other databases,
Cpe FolD is annotated as bifunctional FolD. Modelling and
in vitro studies of Eco FolD predicted that Y50, K54, Q98,
D121 and G122 are involved in THF binding/catalysis
(Sah & Varshney, 2015). Sequence analysis of Cpe FolD
showed that the counterparts of Eco FolD K54, Q98 and
G122 are represented by Q, L and C, respectively, in Cpe FolD. As Cpe FolD possesses substitutions at these key positions, we tested for the biochemical activities of Cpe FolD
and found that it is indeed a monofunctional FolD with
dehydrogenase activity. To test this in vivo, we expressed
Cpe FolD in the E. coli KL16 strain deleted for folD
(DfolD/pCpe Fhs), where it failed to rescue the strain for
its formate and glycine auxotrophies. In previous studies,
it was reported that when K54 of Eco FolD and its counterpart (K56) in the dehydrogenase/cyclohydrolase domains
of the human trifunctional enzyme (DC301) were mutated
to Q or S they lost their cyclohydrolase activities (Sah et al.,
2015; Schmidt et al., 2000). Similarly, mutation of Q100
(counterpart of Q98 of Eco FolD) of DC301 to A, N,
E, K or M showed a loss in cyclohydrolase activity
(Sundararajan & MacKenzie, 2002). The monofunctional
NAD-dependent 5,10-CH2-THF dehydrogenase (yMTD)
from Saccharomyces cerevisiae possesses T57 and Y98 as
the counterparts of K54 and Q98 of Eco FolD. In previous
studies, single mutants T57K and Y98Q, and double
mutant T57K/Y98Q were generated to test the hypothesis
that the lack of cyclohydrolase activity in yMTD was due
to the substitution of a conserved K/Q pair. Although the
mutants retained dehydrogenase activities, they did not
gain cyclohydrolase activity (Wagner et al., 2005). This
suggests that in addition to the K/Q pair, other residues
are also involved in maintaining the proper orientation
of the K/Q pair with 5,10-CH+-THF.
Determination of kinetic constants (Km and Vmax) of Cpe FolD revealed that its Km for NADP+ was similar to that
reported for C. cylindrosporum FolD (Ccy FolD). However,
its Km for 5,10-CH2-THF was about five times higher than
that of Ccy FolD. The Vmax of Ccy FolD for 5,10-CH2-THF
and NADP+ were not reported (Uyeda & Rabinowitz,
1967). However, when compared with the corresponding
values for Eco FolD of Km of 558 and 187 mM, respectively,
and Vmax of 19 and 16 mmol min21 mg21, respectively, for
the two substrates (Sah & Varshney, 2015), Cpe FolD exhibited lower values of Km but higher values of Vmax for both
substrates, suggesting that Cpe FolD is a more efficient dehydrogenase. Also, as shown in this study, the specific activity
of the dehydrogenase for Cpe FolD is about five times higher
than that of the dehydrogenase activity of Eco FolD (Fig. 3a).
However, the biochemical characterization of Cpe FchA
showed that it is a monofunctional cyclohydrolase. The
specific activity of Cpe FchA for 5,10-CH+-THF was about
twofold lower than that of C. formicoaceticum FchA.
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S. Aluri and others
However, the Km of Cpe FchA was slightly less (Clark &
Ljungdahl, 1982). Interestingly, the specific activity of Cpe FchA was *10 times higher than that of the cyclohydrolase
activity of Eco FolD (Fig. 3b).
The Km of 330 mM for (6R,S)-THF or 165 mM for (6S)THF (assuming it to be 50 % of the total) obtained for
Cpe Fhs is about twofold lower than the value reported
for Ccy Fhs [(6S)-THF 370 mM] (Curthoys & Rabinowitz,
1972). Similarly, the Km values of 91 mM for ATP and
3 mM for formate obtained for Cpe Fhs were also lower
than those reported (220 and 8.3 mM, respectively) for
Ccy Fhs (Curthoys & Rabinowitz, 1972).
Consistent with the monofunctional activities of Cpe FolD
and Cpe FchA, introduction of both Cpe-folD and CpefchA together (pCpe FchA-FolD) was required to rescue the
DfolD/pCpe Fhs strain for its deficiencies of formate and glycine (Fig. 6). Furthermore, we were successful in generating
a folD deletion in E. coli with the sole support of pCpe FchAFolD (Figs S4 and 7). pCpe FchA-FolD was also able to
rescue the strain for its TMP hypersensitivity (Fig. 8). The
results presented also demonstrate that both pCpe FchAFolD and pCpe FolD rescue the photorepair defect in the
DfolD strain (Fig. 9). These studies show that a bifunctional
FolD could be substituted for by the two proteins possessing
independent dehydrogenase and cyclohydrolase activities.
The work also sheds light on why FolD, which provides
for the important dehydrogenase and cyclohydrolase activities, is ubiquitous and Fhs is always present in addition to
FolD. Evolutionarily, an efficient cyclohydrolase activity of
FchA in C. perfringens might have paved the way for the
loss of cyclohydrolase activity in Cpe FolD.
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ACKNOWLEDGEMENTS
We thank our laboratory colleagues for their suggestions on the
manuscript. This work was supported by grants from the Department
of Science and Technology (DST) and the Department of Biotechnology (DBT), New Delhi. U. V. is a J. C. Bose fellow of DST, New Delhi.
S. A. was supported by a Senior Research Fellowship of the Council of
Scientific and Industrial Research, New Delhi. S. S. is supported by a
Research Associateship of Dr D. S. Kothari of the University Grants
Commission, New Delhi.
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