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Sergey A. Krupenko – One of the best experts on this subject based on the ideXlab platform.

  • Cytosolic 10-Formyltetrahydrofolate dehydrogenase regulates glycine metabolism in mouse liver
    Scientific Reports, 2019
    Co-Authors: Natalia I. Krupenko, Jaspreet Sharma, Peter Pediaditakis, Baharan Fekry, Kristi L. Helke, Xiuxia Du, Susan Sumner, Sergey A. Krupenko
    Abstract:

    ALDH1L1 (10-Formyltetrahydrofolate dehydrogenase), an enzyme of folate metametabolism highly expressed in liver, metabolizes 10-Formyltetrahydrofolate to produce tetrahydrofolate (THF). This reaction might have a regulatory function towards reduced folate pools, de novo purine biosynthesis, and the flux of folate-bound methyl groups. To understand the role of the enzyme in cellular metametabolism, Aldh1l1 ^−/− mice were generated using an ES cell clone (C57BL/6N background) from KOMP repository. Though Aldh1l1 ^−/− mice were viable and did not have an apparent phenotype, metabolomic analysis indicated that they had metabolic signs of folate deficiency. Specifically, the intermediate of the histidine degradation pathway and a marker of folate deficiency, formiminoglutamate, was increased more than 15-fold in livers of Aldh1l1 ^−/− mice. At the same time, blood folate levels were not changed and the total folate pool in the liver was decreased by only 20%. A two-fold decrease in glycine and a strong drop in glycine conjugates, a likely result of glycine shortage, were also observed in Aldh1l1 ^−/− mice. Our study indicates that in the absence of ALDH1L1 enzyme, 10-formyl-THF cannot be efficiently metabolized in the liver. This leads to the decrease in THF causing reduced generation of glycine from serine and impaired histidine degradation, two pathways strictly dependent on THF.

  • Impact of Aldh1l1 Knockout On Metabolic Phenotype in Mouse Liver
    The FASEB Journal, 2019
    Co-Authors: Jaspreet Sharma, Natalia I. Krupenko, Sergey A. Krupenko
    Abstract:

    ALDH1L1 (10-Formyltetrahydrofolate dehydrogenase), an enzyme of folate metametabolism, is highly expressed in liver. This enzyme metabolizes 10-Formyltetrahydrofolate to produce tetrahydrofolate (THF) …

  • Enzymatic properties of ALDH1L2, a mitochondrial 10-Formyltetrahydrofolate dehydrogenase.
    Chemico-biological interactions, 2011
    Co-Authors: Kyle C. Strickland, Yaroslav Tsybovsky, Natalia I. Krupenko, Marianne E. Dubard, Sergey A. Krupenko
    Abstract:

    10-Formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1), an abundant cytosolic enzyme of folate metametabolism, shares significant sequence similarity with enzymes of the aldehyde dehydrogenase (ALDH) family. The enzyme converts 10-Formyltetrahydrofolate (10-fTHF) to tetrahydrofolate and CO(2) in an NADP(+)-dependent manner. The mechanism of this reaction includes three consecutive steps with the final occurring in an ALDH-homologous domain. We have recently identified a mitochondrial isoform of FDH (mtFDH), which is the product of a separate gene, ALDH1L2. Its overall identity to cytosolic FDH is about 74%, and the identity between the ALDH domains rises up to 79%. In the present study, human mtFDH was expressed in Escherichia coli, purified to homogeneity, and characterized. While the recombinant enzyme was capable of catalyzing the 10-fTHF hydrolase reaction, it did not produce detectable levels of ALDH activity. Despite the lack of typical ALDH catalysis, mtFDH was able to perform the characteristic 10-fTHF dehydrogenase reaction after reactivation by recombinant 4′-phosphopantetheinyl transferase (PPT) in the presence of coenzyme A. Using site-directed mutamutagenesis, it was determined that PPT modifies mtFDH specifically at Ser375. The C-terminal domain of mtFDH (residues 413-923) was also expressed in E. coli and characterized. This domain was found to exist as a tetramer and to catalyze an esterase reaction that is typical of other ALDH enzymes. Taken together, our studies suggest that ALDH1L2 has enzymatic properties similar to its cytosolic counterpart, although the inability to catalyze the ALDH reaction with short-chain aldehyde substrates remains an unresolved issue at present.

Robert J. Cook – One of the best experts on this subject based on the ideXlab platform.

  • Identification of protein-arginine N-methyltransferase as 10-Formyltetrahydrofolate dehydrogenase
    The Journal of biological chemistry, 1998
    Co-Authors: Sangduk Kim, Robert J. Cook, Gil Hong Park, Won A. Joo, Woon Ki Paik, Kenneth R. Williams
    Abstract:

    S-Adenosylmethionine:proteinarginine N-methyltransferase (EC 2.1.1. 23; protein methylase I) transfers the methyl group of S-adenosyl-L-methionine to an arginine residue of a protein substrate. The homogeneous liver protein methylase I was subjected to tryptic digestion followed by reverse phase high performance liquid chrochromatography (HPLC) separation and either “on-line” mass spectrometric fragmentation or “off-line” Edman sequencing of selected fractions. Data base searching of both the mass spectrometric and Edman sequencing data from several peptides identified the protein methylase as 10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6; Cook, R. J., Lloyd, R. S., and Wagner, C. (1991) J. Biol. Chem. 266, 4965-4973; Swiss accession number). This identification was confirmed by comparative HPLC tryptic peptide mapping and affinity chromatography of the methylase on the 5-formyltetrahydrofolate-Sepharose affinity gel used to purify the dehydrogenase. The purified rat liver methylase had approximately 33% of the 10-Formyltetrahydrofolate dehydrogenase and 36% of the aldehyde dehydrogenase activity as compared with the recombinant dehydrogenase, which also had protein methylase I activity. Polyclonal antibodies against recombinant dehydrogenase reacted with protein methylase I purified either by polyacrylamide gel electrophoresis or 5-formyltetrahydrofolate affinity chromatography. In each instance there was only a single immunoreactive band at a molecular weight of approximately 106,000. Together, these results confirm the co-identity of proteinarginine methyltransferase and 10-Formyltetrahydrofolate dehydrogenase.

  • DOMAIN STRUCTURE OF RAT 10-Formyltetrahydrofolate DEHYDROGENASE : RESOLUTION OF THE AMINO-TERMINAL DOMAIN AS 10-Formyltetrahydrofolate HYDROLASE
    The Journal of biological chemistry, 1997
    Co-Authors: Sergey A. Krupenko, Conrad Wagner, Robert J. Cook
    Abstract:

    We expressed the NH2-terminal domain of the multidomain, multifunctional enzyme, 10-Formyltetrahydrofolate dehydrogenase (FDH), using a baculovirus expression system in insect cells. Expression of the 203-amino acid NH2-terminal domain (residues 1-203), which is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.2), resulted in the appearance of insoluble recombinant protein apparently due to incorrect folding. The longer NH2-terminal recombinant protein (residues 1-310), which shares 32% identity with Escherichia coli L-methionyl-tRNA formyltransferase (EC 2.1.2.9), was expressed as a soluble protein. During expression, this protein was released from cells to the culture medium and was purified from the culture medium by 5-formyltetrahydrofolate-Sepharose affinity chromatography followed by chromatography on a Mono-Q column. We found that the purified NH2-terminal domain bears a folate binding site, possesses 10-Formyltetrahydrofolate hydrolase activity, and exists as a monomer. Titration of tryptophan fluorescence showed that native FDH bound both the substrate of the reaction, 10-formyl-5, 8-dideazafolate, and the product of the reaction, 5,8-dideazafolate, with the same affinities as its NH2-terminal domain did and that both proteins bound the substrate with a 50-fold higher affinity than the product. Neither the NH2-terminal domain nor its mixture with the previously purified COOH-terminal domain had 10-Formyltetrahydrofolate dehydrogenase activity. Formation of complexes between the COOH- and NH2-terminal domains also was not observed. We conclude that the 10-Formyltetrahydrofolate dehydrogenase activity of FDH is a result of the action of the aldehyde dehydrogenase catalytic center residing in the COOH-terminal domain on the substrate bound in the NH2-terminal domain and that the intermediate domain is necessary to bring the two functional domains together in the correct orientation.

  • Expression, Purification, and Properties of the Aldehyde Dehydrogenase Homologous Carboxyl-terminal Domain of Rat 10-Formyltetrahydrofolate Dehydrogenase
    The Journal of biological chemistry, 1997
    Co-Authors: Sergey A. Krupenko, Conrad Wagner, Robert J. Cook
    Abstract:

    Abstract The liver cytosolic enzyme, 10-Formyltetrahydrofolate dehydrogenase (FDH) (EC 1.5.1.6) catalyzes two reactions: the NADP+-dependent oxidation of 10-Formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP+-independent hydrolysis of 10-Formyltetrahydrofolate to tetrahydrofolate and formate. The COOH-terminal domain of the enzyme (residues 420-902) is about 48% identical to a family of NAD-dependent aldehyde dehydrogenases (EC 1.2.1.3), and FDH possesses aldehyde dehydrogenase activity. We expressed the COOH-terminal domain (residues 420-902) of FDH in insect cells using a baculovirus expression system. The recombinant protein was released from insect cells to the culture medium and was purified from the medium by a two-step procedure: precipitation with 35% saturated ammonium sulfate followed by chromatography on hydroxyapatite. The purified COOH-terminal domain displayed aldehyde dehydrogenase activity similar to that of native FDH but had neither dehydrogenase nor hydrolase activity toward folate substrates. Aldehyde dehydrogenase activity of the COOH-terminal domain and FDH was independent of the presence of 2-mercaptoethanol while 10-FDDF dehydrogenase activity of FDH occurred only in the presence of 2-mercaptoethanol. The COOH-terminal domain existed as a tetramer showing that the sites for oligomerization of subunits in native FDH resides in this domain. Using titration of tryptophan fluorescence, it was found that the COOH-terminal domain bound NADP+ to the same extent as FDH (Kd 0.2 and 0.3 μM, respectively) but did not bind folate. Both FDH and its COOH-terminal domain also bound NAD+ (Kd 11 and 16 μM, respectively) as measured by fluorescence titration. Both proteins were able to catalyze the aldehyde dehydrogenase reaction utilizing NADP+ or NAD+, but the Km for NAD+ was three orders higher than that for NADP+ (2 mM and 1.5-2.0 μM, respectively). The concentration of NAD+ required for the reaction was high compared with the physiological level of NAD+, suggesting that the reaction does not occur in vivo NAD+ at physiological concentrations stimulated the aldehyde dehydrogenase reaction performed by FDH or its COOH-terminal domain using NADP+.

Conrad Wagner – One of the best experts on this subject based on the ideXlab platform.

  • On the role of conserved histidine 106 in 10-Formyltetrahydrofolate dehydrogenase catalysis: connection between hydrolase and dehydrogenase mechanisms.
    The Journal of biological chemistry, 2001
    Co-Authors: Sergey A. Krupenko, Alexander P. Vlasov, Conrad Wagner
    Abstract:

    The enzyme, 10-Formyltetrahydrofolate dehydrogenase (FDH), converts 10-Formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate in an NADP+-dependent dehydrogenase reaction or an NADP+-independent hydrolase reaction. The hydrolase reaction occurs in a 310-amino acid long amino-terminal domain of FDH (Nt-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. The amino-terminal domain of FDH shares some sequence identity with several other enzymes utilizing 10-formyl-THF as a substrate. These enzymes have two strictly conserved residues, aspartate and histidine, in the putative catalytic center. We have shown recently that the conserved aspartate is involved in FDH catalysis. In the present work we studied the role of the conserved histidine, His106, in FDH function. Site-directed mutamutagenesis experiments showed that replacement of the histidine with alanine, asparagine, aspartate, glutamate, glutamine, or arginine in Nt-FDH resulted in expression of insoluble proteins. Replacement of the histidine with another positively charged residue, lysine, produced a soluble mutant with no hydrolase activity. The insoluble mutants refolded from inclusion bodies adopted a conformation inherent to the wild-type Nt-FDH, but they did not exhibit any hydrolase activity. Substitution of alanine for three non-conserved histidines located close to the conserved one did not reveal any significant changes in the hydrolase activity of Nt-FDH. Expressed full-length FDH with the substitution of lysine for the His106 completely lost both the hydrolase and dehydrogenase activities. Thus, our study showed that His106, besides being an important structural residue, is also directly involved in both the hydrolase and dehydrogenase mechanisms of FDH. Modeling of the putative hydrolase catalytic center/folate-binding site suggested that the catalytic residues, aspartate and histidine, are unlikely to be adjacent to the catalytic cysteine in the aldehyde dehydrogenase catalytic center. We hypothesize that 10-formyl-THF dehydrogenase reaction is not an independent reaction but is a combination of hydrolase and aldehyde dehydrogenase reactions.

  • Aspartate 142 Is Involved in Both Hydrolase and Dehydrogenase Catalytic Centers of 10-Formyltetrahydrofolate Dehydrogenase
    The Journal of biological chemistry, 1999
    Co-Authors: Sergey A. Krupenko, Conrad Wagner
    Abstract:

    Abstract The enzyme 10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes conversion of 10-Formyltetrahydrofolate to tetrahydrofolate in either a dehydrogenase or hydrolase reaction. The hydrolase reaction occurs in a 310-residue amino-terminal domain of FDH (Nt-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. Nt-FDH shares some sequence identity with several 10-Formyltetrahydrofolate-utilizing enzymes. All these enzymes have a strictly conserved aspartate, which is Asp142 in the case of Nt-FDH. Replacement of the aspartate with alanine, asparagine, glutamate, or glutamine in Nt-FDH resulted in complete loss of hydrolase activity. All the mutants, however, were able to bind folate, although with lower affinity than wild-type Nt-FDH. Six other aspartate residues located near the conserved Asp142 were substituted with an alanine, and these substitutions did not result in any significant changes in the hydrolase activity. The expressed D142A mutant of the full-length enzyme completely lost both hydrolase and dehydrogenase activities. This study shows that Asp142 is an essential residue in the enzyme mechanism for both the hydrolase and dehydrogenase reactions of FDH, suggesting that either the two catalytic centers of FDH are overlapped or the dehydrogenase reaction occurs within the hydrolase catalytic center.

  • Overexpression of functional hydrolase domain of rat liver 10-Formyltetrahydrofolate dehydrogenase in Escherichia coli.
    Protein expression and purification, 1998
    Co-Authors: Sergey A. Krupenko, Conrad Wagner
    Abstract:

    Rat liver 10-Formyltetrahydrofolate dehydrogenase (FDH) is a tetrameric enzyme composed of four identical 902-amino-acid-residue (99 kDa) monomers. We expressed the enzyme and its 310-amino-acid-residue amino-terminal domain, which is 10-Formyltetrahydrofolate hydrolase, in Escherichia coli BL21 (DE3) cells using the pRSET expression vector. We removed the entire translated region of the vector including the polyhistidyl tag and the recombinant proteins were expressed, not as a fusion constructs, but as unmodified sequences. The expressed full-length enzyme was found to be an insoluble protein and was not purified and characterized, while the amino-terminal domain was expressed as a soluble protein possessing hydrolase activity. The recombinant amino-terminal domain was purified in one step on a DEAE MemSep 1000 HP Ion-Exchange Membrane Chromatography Cartridge (Millipore) using a ConSep LC100 chromatographic system (Millipore). The chromatography gave a homogenous and active preparation of the recombinant protein with a yield of about 2 mg per 100 ml of bacterial culture. Kinetic parameters of the hydrolase reaction displayed by the amino-terminal domain expressed in E. coli were similar to those of the recombinant full-length enzyme and its amino-terminal domain previously expressed in insect cells. The purified recombinant enzyme remained active for at least 4 weeks at 4 degreesC. These results show that the hydrolase amino-terminal domain of FDH can be overexpressed as a functional enzyme in E. coli cells and purified in one step by a simple chromatographic procedure.

Natalia I. Krupenko – One of the best experts on this subject based on the ideXlab platform.

  • Cytosolic 10-Formyltetrahydrofolate dehydrogenase regulates glycine metabolism in mouse liver
    Scientific Reports, 2019
    Co-Authors: Natalia I. Krupenko, Jaspreet Sharma, Peter Pediaditakis, Baharan Fekry, Kristi L. Helke, Xiuxia Du, Susan Sumner, Sergey A. Krupenko
    Abstract:

    ALDH1L1 (10-Formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism highly expressed in liver, metabolizes 10-Formyltetrahydrofolate to produce tetrahydrofolate (THF). This reaction might have a regulatory function towards reduced folate pools, de novo purine biosynthesis, and the flux of folate-bound methyl groups. To understand the role of the enzyme in cellular metabolism, Aldh1l1 ^−/− mice were generated using an ES cell clone (C57BL/6N background) from KOMP repository. Though Aldh1l1 ^−/− mice were viable and did not have an apparent phenotype, metabolomic analysis indicated that they had metabolic signs of folate deficiency. Specifically, the intermediate of the histidine degradation pathway and a marker of folate deficiency, formiminoglutamate, was increased more than 15-fold in livers of Aldh1l1 ^−/− mice. At the same time, blood folate levels were not changed and the total folate pool in the liver was decreased by only 20%. A two-fold decrease in glycine and a strong drop in glycine conjugates, a likely result of glycine shortage, were also observed in Aldh1l1 ^−/− mice. Our study indicates that in the absence of ALDH1L1 enzyme, 10-formyl-THF cannot be efficiently metabolized in the liver. This leads to the decrease in THF causing reduced generation of glycine from serine and impaired histidine degradation, two pathways strictly dependent on THF.

  • Impact of Aldh1l1 Knockout On Metabolic Phenotype in Mouse Liver
    The FASEB Journal, 2019
    Co-Authors: Jaspreet Sharma, Natalia I. Krupenko, Sergey A. Krupenko
    Abstract:

    ALDH1L1 (10-Formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism, is highly expressed in liver. This enzyme metabolizes 10-Formyltetrahydrofolate to produce tetrahydrofolate (THF) …

  • Enzymatic properties of ALDH1L2, a mitochondrial 10-Formyltetrahydrofolate dehydrogenase.
    Chemico-biological interactions, 2011
    Co-Authors: Kyle C. Strickland, Yaroslav Tsybovsky, Natalia I. Krupenko, Marianne E. Dubard, Sergey A. Krupenko
    Abstract:

    10-Formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1), an abundant cytosolic enzyme of folate metabolism, shares significant sequence similarity with enzymes of the aldehyde dehydrogenase (ALDH) family. The enzyme converts 10-Formyltetrahydrofolate (10-fTHF) to tetrahydrofolate and CO(2) in an NADP(+)-dependent manner. The mechanism of this reaction includes three consecutive steps with the final occurring in an ALDH-homologous domain. We have recently identified a mitochondrial isoform of FDH (mtFDH), which is the product of a separate gene, ALDH1L2. Its overall identity to cytosolic FDH is about 74%, and the identity between the ALDH domains rises up to 79%. In the present study, human mtFDH was expressed in Escherichia coli, purified to homogeneity, and characterized. While the recombinant enzyme was capable of catalyzing the 10-fTHF hydrolase reaction, it did not produce detectable levels of ALDH activity. Despite the lack of typical ALDH catalysis, mtFDH was able to perform the characteristic 10-fTHF dehydrogenase reaction after reactivation by recombinant 4′-phosphopantetheinyl transferase (PPT) in the presence of coenzyme A. Using site-directed mutagenesis, it was determined that PPT modifies mtFDH specifically at Ser375. The C-terminal domain of mtFDH (residues 413-923) was also expressed in E. coli and characterized. This domain was found to exist as a tetramer and to catalyze an esterase reaction that is typical of other ALDH enzymes. Taken together, our studies suggest that ALDH1L2 has enzymatic properties similar to its cytosolic counterpart, although the inability to catalyze the ALDH reaction with short-chain aldehyde substrates remains an unresolved issue at present.

Yaroslav Tsybovsky – One of the best experts on this subject based on the ideXlab platform.

  • Enzymatic properties of ALDH1L2, a mitochondrial 10-Formyltetrahydrofolate dehydrogenase.
    Chemico-biological interactions, 2011
    Co-Authors: Kyle C. Strickland, Yaroslav Tsybovsky, Natalia I. Krupenko, Marianne E. Dubard, Sergey A. Krupenko
    Abstract:

    10-Formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1), an abundant cytosolic enzyme of folate metabolism, shares significant sequence similarity with enzymes of the aldehyde dehydrogenase (ALDH) family. The enzyme converts 10-Formyltetrahydrofolate (10-fTHF) to tetrahydrofolate and CO(2) in an NADP(+)-dependent manner. The mechanism of this reaction includes three consecutive steps with the final occurring in an ALDH-homologous domain. We have recently identified a mitochondrial isoform of FDH (mtFDH), which is the product of a separate gene, ALDH1L2. Its overall identity to cytosolic FDH is about 74%, and the identity between the ALDH domains rises up to 79%. In the present study, human mtFDH was expressed in Escherichia coli, purified to homogeneity, and characterized. While the recombinant enzyme was capable of catalyzing the 10-fTHF hydrolase reaction, it did not produce detectable levels of ALDH activity. Despite the lack of typical ALDH catalysis, mtFDH was able to perform the characteristic 10-fTHF dehydrogenase reaction after reactivation by recombinant 4′-phosphopantetheinyl transferase (PPT) in the presence of coenzyme A. Using site-directed mutagenesis, it was determined that PPT modifies mtFDH specifically at Ser375. The C-terminal domain of mtFDH (residues 413-923) was also expressed in E. coli and characterized. This domain was found to exist as a tetramer and to catalyze an esterase reaction that is typical of other ALDH enzymes. Taken together, our studies suggest that ALDH1L2 has enzymatic properties similar to its cytosolic counterpart, although the inability to catalyze the ALDH reaction with short-chain aldehyde substrates remains an unresolved issue at present.

  • 10 formyltetrahydrofolate dehydrogenase requires a 4 phosphopantetheine prosthetic group for catalysis
    Journal of Biological Chemistry, 2007
    Co-Authors: Henry Donato, Natalia I. Krupenko, Yaroslav Tsybovsky, Sergey A. Krupenko
    Abstract:

    Abstract 10-Formyltetrahydrofolate dehydrogenase (FDH) consists of two independent catalytic domains, N- and C-terminal, connected by a 100-amino acid residue linker (intermediate domain). Our previous studies on structural organization and enzymatic properties of rat FDH suggest that the overall enzyme reaction, i.e. NADP+-dependent conversion of 10-Formyltetrahydrofolate to tetrahydrofolate and CO2, consists of two steps: (i) hydrolytic cleavage of the formyl group in the N-terminal catalytic domain, followed by (ii) NADP+-dependent oxidation of the formyl group to CO2 in the C-terminal aldehyde dehydrogenase domain. In this mechanism, it was not clear how the formyl group is transferred between the two catalytic domains after the first step. This study demonstrates that the intermediate domain functions similarly to an acyl carrier protprotein. A 4′-phosphopantetheine swinging arm bound through a phosphoester bond to Ser354 of the intermediate domain transfers the formyl group between the catalytic domains of FDH. Thus, our study defines the intermediate domain of FDH as a novel carrier protprotein and provides the previously lacking component of the FDH catalytic mechanism.

  • 10-Formyltetrahydrofolate Dehydrogenase Requires a 4′-Phosphopantetheine Prosthetic Group for Catalysis
    Journal of Biological Chemistry, 2007
    Co-Authors: Henry Donato, Natalia I. Krupenko, Yaroslav Tsybovsky, Sergey A. Krupenko
    Abstract:

    Abstract 10-Formyltetrahydrofolate dehydrogenase (FDH) consists of two independent catalytic domains, N- and C-terminal, connected by a 100-amino acid residue linker (intermediate domain). Our previous studies on structural organization and enzymatic properties of rat FDH suggest that the overall enzyme reaction, i.e. NADP+-dependent conversion of 10-Formyltetrahydrofolate to tetrahydrofolate and CO2, consists of two steps: (i) hydrolytic cleavage of the formyl group in the N-terminal catalytic domain, followed by (ii) NADP+-dependent oxidation of the formyl group to CO2 in the C-terminal aldehyde dehydrogenase domain. In this mechanism, it was not clear how the formyl group is transferred between the two catalytic domains after the first step. This study demonstrates that the intermediate domain functions similarly to an acyl carrier protprotein. A 4′-phosphopantetheine swinging arm bound through a phosphoester bond to Ser354 of the intermediate domain transfers the formyl group between the catalytic domains of FDH. Thus, our study defines the intermediate domain of FDH as a novel carrier protprotein and provides the previously lacking component of the FDH catalytic mechanism.