Chemicals and reagents
All chemicals used in the study were analytical grade. L-serine, NADPH, NADP+, PLP, polyethyleneimine (PEI) solution (50% w/v), D-glucose, N-Z-amine AS (casein enzymatic hydrolysate), thiosemicarbazide, and α-lactose were purchased from Sigma-Aldrich (St Louis, MO, USA). [6R,S] THF, [6 S] THF, and [6R] 5,10-CH2-THF were obtained from Merck Eprova AG (Schaffhausen Switzerland). D-cycloserine, dithiothreitol (DTT) and yeast extract were from Bio-Science Inc. (Allentown, PA, USA). Isopropyl thio-β-D-galactoside (IPTG) was purchased from Fermentas Life Sciences (Glen Burnie, MD, USA). All chromatographic media were purchased from GE Healthcare Biosciences (Uppsala, Sweden). N-(2-hydroxyethyl) piperazine-N’-(2-ethane-sulfonic acid) (HEPES) was purchased from Research Organics (Cleveland, OH, USA). Escherichia coli BL21 (DE3) (Novagen, Madison, WI, USA) was used as the host strain for protein expression.
Protein expression and purification
Two expression media types, LB-IPTG and auto-induction media were used to express the recombinant Plasmodium SHMTs in an E. coli system. Protein expression of Pf- and PvSHMT using LB-IPTG media was performed according to previous reports [6, 7]. The auto-induction media used was modified from the standard formula previously described . Briefly, a starter culture was grown at 37°C overnight in ZYP-0.8G media (1% w/v N-Z-amine AS, 0.5% w/v yeast extract, 62.5 mM (NH4)2SO4, 125 mM KH2PO4, 125 mM Na2HPO4, 1 mM MgSO4, and 0.8% w/v D-glucose) supplemented with 50 μg/ml ampicillin. The starter culture (0.5% v/v) was inoculated in ZYP-5052 media (1% w/v N-Z-amine AS, 0.5% w/v yeast extract, 0.5% w/v glycerol, 0.2% w/v α-lactose, and 0.05% w/v glucose) containing 50 μg/ml ampicillin, and the culture was vigorously shaken at 37°C until the OD600 reached ~1.0 (6–7 hours). The temperature was lowered to 16°C, and the cells were incubated at this temperature for 16–18 hours before they were harvested. Protein purification was carried out according to the procedures previously described [6, 7], except that only a Ni-Sepharose column was used for PfSHMT purification. For long-term storage at −80°C, the purified PvSHMT was kept in 50 mM HEPES, pH 7 containing 0.5 mM EDTA and 1 mM DTT (Buffer A), and PfSHMT was kept in Buffer A with 10% v/v glycerol added (Buffer B). Unless otherwise indicated, biochemical studies of Pf- and PvSHMT were performed in Buffer A.
The expression and purification of E. coli MTHFD was performed as described in  with some modifications. Briefly, BL21DE3 carrying pET22b(+)::FolD was grown at 37°C until OD600 reached 1.2, at which IPTG was added to 0.4 mM. Cells were cultured until OD600 reached 5 before harvesting. Cell pellet was re-suspended in 50 mM potassium phosphate buffer pH 6.5, 1 mM DTT, 1 mM EDTA and 0.1 mM PMSF, and lysed by ultrasonication (Sonic Vibra cellTM; model VCX750). MTHFD was precipitated using 0-30% ammonium sulfate and the protein precipitation was dissolved in 50 mM potassium phosphate buffer pH 6.5, 1 mM DTT, 0.3 mM EDTA (buffer C). The dissolved protein was dialyzed against buffer C and loaded onto a DEAE-column previously equilibrated with the same buffer. Proteins were eluted with a linear gradient of 0–300 mM NaCl in buffer C. The activity of MTHFD was determined spectrophotometrically by monitoring the increase in absorbance at 375 nm due to the formation of NADPH by the oxidation of 5,10-CH2-THF. The purified MTHFD stored at −80°C was stable for at least three months.
The concentration of proteins was determined by the Bradford method  using the standard dye reagent (Bio-Rad Life Science, CA, USA). The protein concentration was calculated from a standard curve using bovine serum albumin as a protein standard. Alternatively, protein concentrations were determined according to the enzyme UV-visible absorption using absorption coefficient values at 420 (5,400 M-1 cm-1), 422 nm (6,370 M-1 cm-1), and 280 nm (14,690 M-1 cm-1) for PfSHMT, PvSHMT, and MTHFD respectively [6, 7]. The MTHFD absorption coefficient was calculated based on the primary amino acid sequence .
SHMT activity assay
To monitor Plasmodium SHMT activity during enzyme preparation, the SHMT reaction was coupled with a MTHFD reaction (SHMT-MTHFD) and performed under regular aerobic conditions in Buffer A. A typical assay reaction contained 5 μM MTHFD, 2 mM L-serine, 0.4 mM THF, 0.25 mM NADP+, and SHMT in a final volume of 1 mL at 25°C. Progression of the reaction was monitored by an increase in absorbance at 375 nm. Measurement of steady-state kinetic parameters of Plasmodium SHMTs was performed using the MTHFD coupled assay with a rapid-mixing apparatus (SFA-20, TgK Scientific, Bradford-on-Avon, UK) connected to a double-beam spectrophotometer (SHIMADZU 2501 PC, Shimadzu corp., Kyoto, Japan). To prolong the stability of THF, a stock solution of THF was prepared in an anaerobic glove box. The apparent Michaelis constant (Kmapp) for THF was determined by fixing the concentration of L-serine at 2 mM and varying the concentration of THF between 0.025-0.4 mM. A similar set-up was used in determining Kmapp for L-serine, except that the concentration of THF was fixed at 0.4 mM and the concentrations of L-serine were varied between 0.05-1.6 mM. All concentrations indicated were final concentrations after mixing.
Inhibitor screening for
Inhibition of SHMT was studied by measuring the initial rates of the reaction using the SHMT-MTHFD coupling system, as described in “SHMT activity assay” of the Methods section, in the presence of inhibitors. Inhibitors used in this study were anti-folates (2,4-diaminopyrimidine) and amino acid analogues (D-serine, D-alanine, D-threonine, L-allo-threonine, D-cycloserine and thiosemicarbazide). Stock solutions of anti-folates were prepared in absolute dimethyl sulfoxide (DMSO) and amino acid analogues were prepared in Buffer A. The final concentrations used for anti-folates were 0.05-0.5 mM, depending on the solubility of each compound. The final concentration for the amino acid analogues was 1 mM. The efficacy of the inhibitors is presented as % inhibition, which is a relative percentage of enzyme activity compared to the reaction in the absence of the inhibitor.
SHMT inactivation by thiosemicarbazide
Inactivation of Pf- and PvSHMT by thiosemicarbazide was investigated by monitoring the residual SHMT activity upon incubation of the enzyme with various thiosemicarbazide concentrations at various incubation times using a rapid-mixing apparatus connected to a double-beam spectrophotometer. One syringe of the rapid-mixing apparatus contained 1 μM Pf- or PvSHMT, 5 μM MTHFD and various thiosemicarbazide concentrations (0.03-1 mM). Another syringe contained 2 mM L-serine, 0.4 mM THF and 0.25 mM NADP+. All reactions were performed in Buffer A at 25°C and the reaction was initiated by mixing the solutions from both syringes. Time-dependent inactivation was performed by varying the incubation time (5–30 min) of enzyme with thiosemicarbazide in the first syringe before mixing with the solution in the second syringe.
The inactivation reaction appeared to follow first-order kinetics since a plot of ln V
time was linear. V
represent initial velocities of the reaction in the presence and the absence of inhibitor, respectively. An observed rate constant (kobs
) at each thiosemicarbazide concentration was determined from a slope of the plot of ln V
incubation time. A rate constant for the inactivation step (kinact
) and the equilibrium dissociation constant for binding of the inhibitor (KI
) were calculated from Equation 1
, where [I] is the concentration of the inhibitor, using non-linear algorithms found in KaleidaGraph software (Synergy Software, Reading, PA, USA).
Analysis of product from the inactivation of PvSHMT by thiosemicarbazide
The product that resulted from the inactivation of PvSHMT by thiosemicarbazide was analysed by UV-visible absorption, retention time analysis after HPLC separation, and molecular mass determination by LC-MS. PvSHMT with OD422 ~ 0.4 AU (62.79 μM) was incubated with 10 mM thiosemicarbazide for 50 min in Buffer A at 25 °C, and the absorption spectrum change was recorded. The enzyme was de-natured by adding SDS (final concentration of 1% w/v). The de-natured enzyme was separated from small molecular weight compounds by a Centricon device with a 10 kDa molecular weight cut-off membrane (Millipore, Carrigtwohill, Co. Cork, Ireland), and the spectrum of the filtrate was recorded.
The filtrate from ultrafiltration of the PvSHMT-thiosemicarbazide mixture was subjected to reverse phase HPLC chromatography (Polaris 3 C8-A, 50 x 4.6 mm; Agilent Technologies, Inc. Santa Clara, CA, USA). The column was pre-equilibrated with 25 mM sodium formate pH 4.3 and was eluted using the same buffer at a flow rate of 1 mL min-1. The eluted compounds were detected by UV-visible absorption.
Additionally, the filtrate was analysed by LC-MS (Bruker AXS Inc., Madison, WI, USA) to separate small molecules using a Polaris 3 C8-A column pre-equilibrated with 25 mM ammonium formate pH 6.5 at a flow rate of 0.5 mL min-1 at 25°C. Eluents were analysed for their masses using a linear ion trap MS equipped with an electrospray ionization (ESI) source. The parental and fragmented mass profiles were analysed. All buffers used were pre-filtered through a 0.45 mm membrane (Millipore, Carrigtwohill, Co, Cork, Ireland).
Similar experiments as described above were applied for free PLP (OD388 ~ 0.1 AU) in the presence of 10 mM thiosemicarbazide.
Fluorescence changes of
SHMTs upon binding of amino acids
Changes in the fluorescence properties of Pf- and PvSHMT upon binding of amino acids and folate analogues were monitored using a spectrofluorophotometer (SHIMADZU RF5301 PC, Shimadzu corp., Kyoto, Japan) at 25°C. The emission and excitation monochromator slits were set at 5 nm, the light source was from xenon lamp (150 W), and the scanning rate was set at medium speed. The concentrations of free PLP, Pf- and PvSHMT were ~ 23 μM (PLP; OD388 ~ 0.12, PfSHMT; OD420 ~ 0.12, and PvSHMT; OD422 ~ 0.15). Free PLP, Pf- and PvSHMT were excited at the wavelengths 388, 420 and 422 nm, respectively. L-serine, D-serine, L-alanine, or glycine was added to the protein or PLP in Buffer A (at the above concentrations) to give a final amino acid concentration of 10 mM, except for folinic acid, which was added to a final concentration of 1 mM. The binding of folinic acid to Plasmodium SHMTs was performed in the absence and presence of 10 mM glycine. For the measurement performed in the presence of both ligands, the enzyme was incubated with glycine for 5 min prior to the addition of folinic acid.