Acyl carrier protein-specific 4'-phosphopantetheinyl transferase activates 10-formyltetrahydrofolate dehydrogenase

Abstract

4'-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4'-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G(1) arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions.

FIGURE 1.

Domain structure of FDH and catalytic activities of apo- and holoenzyme. The amino-terminal domain of FDH catalyzes the hydrolysis of 10-fTHF to THF and formate; the carboxyl-terminal aldehyde dehydrogenase domain catalyzes the oxidation of short chain aldehydes to their respective carboxylic acids. 10-fTHF dehydrogenase catalysis requires the full-length holoenzyme, including the 4-PP prosthetic group covalently attached to serine 354 of the carrier protein-related intermediate domain.

FIGURE 2.

Activation of FDH by purified recombinant human PPT. A, SDS-PAGE (Coomassie-stained) of recombinant human PPT (His₆-tagged, expressed in E. coli) purified by metal affinity chromatography. B, time dependence of apo-FDH activation by the purified PPT (1.4 μm). 5 μm apo-FDH (purified recombinant enzyme expressed in E. coli) was used for each time point. C, activation of apo-FDH by different concentrations of PPT. 4 μm apo-FDH was incubated for 1 min with increasing concentrations of PPT. In all experiments, 100 μm CoA was used. Activation of FDH was measured by reconstituted 10-fTHF dehydrogenase activity as described under “Experimental Procedures.”

FIGURE 3.

Fluorescent labeling of apo-FDH by recombinant PPT. A, SDS-PAGE of recombinant apo-FDH incubated with recombinant PPT and fluorescent reporter for different periods of time. Upper panel, fluorescence of the gel after UV irradiation. Lower panel, Coomassie stain of the same gel. B, quantification of the fluorescence from A. C, fluorescence labeling of wild-type and S354A mutant apo-FDH by recombinant PPT. Upper panel, fluorescence under UV irradiation of SDS-PAGE. Lower panel, Coomassie stain of the same gel.

FIGURE 4.

PPT siRNA silencing prevents apo-FDH fluorescent labeling. A, modification of FDH using a CoA-derived fluorescent reporter was determined after incubation of E. coli-expressed FDH with PPT, water (control), or cell-free extracts from HEK293 or A549 cells. Reaction mixtures were subjected to SDS-PAGE, and the gel was exposed to UV light irradiation (upper panel) and then stained with Coomassie Blue (lower panel). B, levels of PPT mRNA in A549 cells measured by RT-PCR at different times after siRNA transfection. Samples were normalized by the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). C, levels of PPT protein upon siRNA transfection in the same cells detected by Western blotting. The time post-transfection is indicated. Levels of actin are shown as loading control. Time point 0 in B and C shows levels in nontreated cells. D, lysates from A549 cells transfected with PPT-specific (-PPT) or scrambled siRNA (Scr) were incubated with recombinant FDH in the presence of fluorescent reporter and MgCl₂. Fluorescent modification of a His-tagged mutant of FDH (N_t-FDH+IntD), truncated at residue 408, was also evaluated.

FIGURE 5.

Effects of PPT siRNA silencing on A549 cells. A, cell proliferation evaluated by MTT assays. B, cell cycle assessed by FACS analysis of propidium iodide-stained cells 24 h after transfection with scrambled or PPT siRNA. C, apoptosis in PPT siRNA-transfected cells examined by annexin V assays.