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 posttranslational 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 CO 2 . 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.Folate derivatives function as coenzymes in reactions involving the transfer of one-carbon units and participate in key metabolic processes, including nucleotide biosynthesis and the regeneration of methionine from homocysteine (1). The intracellular folate pool consists of several major reduced folate forms, and the overall balance between different folates is maintained by numerous enzymes that incorporate one-carbon groups into the pool, convert these groups within the pool, or utilize them in biosynthetic reactions. One of the folate enzymes, 10-formyltetrahydrofolate (10-fTHF) 2 dehydrogenase (FDH, aldehyde dehydrogenase 1L1, EC 1.5.1.6), oxidizes the formyl group of 10-fTHF to yield tetrahydrofolate (THF) and CO 2 . This reaction requires NADP ϩ and proceeds through a complex multistep mechanism (2). FDH is expressed in most tissues and is particularly abundant in the liver and kidney; in the former it is present at the level of about 1% of the total soluble cytosolic protein (3).The FDH substrate, 10-fTHF, is used as a formyl group donor in two steps of de novo purine biosynthesis (4, 5). The excess of 10-fTHF, not required for this pathway, can be utilized by FDH to replenish the THF pool, thus making folate available for other reactions of one-carbon metabolism. It has been proposed that the FDH reaction serves as a regulator of intracellular purine levels, preventing the excessive flux of one-carbon groups into this pathway (6). Several other metabolic functions have also been attributed to FDH, including storage of intracellular folate in the form of THF (7); detoxification of intracellular formic acid by removing it as CO 2 (8); and regulation of o...

ALDH1L2 Is the Mitochondrial Homolog of 10-Formyltetrahydrofolate Dehydrogenase

“…This has been done by site-directed mutagenesis using a QuikChange kit (Stratagene). The recombinant protein was purified on a metal affinity column and then on a Sephacryl S300 column (100 ϫ 1.5 cm) as described elsewhere (28,29). The sequence of the protein was confirmed by liquid chromatography/tandem mass spectrometry analysis of peptides after tryptic digestion at the MUSC Biomolecular Mass Spectrometry facility.…”

“…The antibody was generated against a 408-amino acid-long N-terminal peptide (residues 23-429, mitochondrial leader sequence was excluded) of ALDH1L2 protein using Harlan Laboratories, Inc. (Indianapolis, IN) services. The truncated mitochondrial FDH was expressed in Escherichia coli as a fusion with His 6 tag at the N terminus according to a procedure we have used in our previous studies (28,29). The expression vector was generated from pRSET/mtFDH plasmid by deleting the mitochondrial leader sequence and introducing an in-frame stop codon immediately downstream of the codon corresponding to Asp-429.…”

Modeling of interactions between functional domains of ALDH1L1

“…While the closed conformation is suggested by the necessity to bring the 4-PP S-formyl moiety in proximity with the dehydrogenase catalytic cysteine (14,19,20,35), the nature of the extended conformation is less obvious. Such an extended conformation, however, is suggested not only by the multicenter ALDH1L1 catalytic mechanisms, but also by the necessity for the ACP domain to interact with 4-PP transferase, the only enzyme in humans that adds the 4-PP to acyl carrier proteins (36,37). For example, the structure of the complex between the human ACP with its phosphopantetheinyl transferase (Fig.…”

Structures of the hydrolase domain of zebrafish 10-formyltetrahydrofolate dehydrogenase and its complexes reveal a complete set of key residues for hydrolysis and product inhibition

“…The structural flexibility at the end of the ligands might facilitate nucleophilic attack on the formyl group and transfer of the formyl group to the intermediate domain of FDH. This domain requires a 4 0 -PP prosthetic group covalently bound to Ser354 in rat Nt-FDH (Ser355 in zNt-FDH) and is proposed to act as a swinging arm to transfer the formyl group to the C-terminal dehydrogenase domain coupling NADP + for the conversion of 10-FTHF to THF and CO 2 (Krupenko, 2009;Strickland et al, 2010). An inspection of the structures of the ligand-free apo zNt-FDH and its respective complexes with 10-FDDF and THF reveals that the catalytic residue His106 remains in a similar position, whereas the positions of Ser108 and Asp142 are shifted towards the formyl group by $0.9 and 0.8 Å , respectively, in the active site as observed in the structure of the THF complex.…”