Enzymatic properties of ALDH1L2, a mitochondrial 10-formyltetrahydrofolate dehydrogenase

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.     

Acyl Carrier Protein-specific 4��-Phosphopantetheinyl Transferase Activates 10-Formyltetrahydrofolate Dehydrogenase

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 G₁ 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.     

Disruption of a calmodulin central helix-like region of 10-formyltetrahydrofolate dehydrogenase impairs its dehydrogenase activity by uncoupling the functional domains

10-Formyltetrahydrofolate dehydrogenase (FDH) is composed of three domains and possesses three catalytic activities but has only two catalytic centers. The amino-terminal domain (residue 1-310) bears 10-formyltetrahydrofolate hydrolase activity, the carboxyl-terminal domain (residue 420-902) bears an aldehyde dehydrogenase activity, and the full-length FDH produces 10-formyltetrahydrofolate dehydrogenase activity. The intermediate linker (residues 311-419) connecting the two catalytic domains does not contribute directly to the enzyme catalytic centers but is crucial for 10-formyltetrahydrofolate dehydrogenase activity. We have identified a region within the intermediate domain (residues 384-405) that shows sequence similarity to the central helix of calmodulin. Deletion of either the entire putative helix or the central part of the helix or replacement of the six residues within the central part with alanines resulted in total loss of the 10-formyltetrahydrofolate dehydrogenase activity, whereas the full hydrolase and aldehyde dehydrogenase activities were retained. Alanine-scanning mutagenesis revealed that neither of the six residues alone is required for FDH activity. Analysis of the predicted secondary structures and circular dichroic and fluorescence spectroscopy studies of the intermediate domain expressed as a separate protein showed that this region is likely to consist of two alpha-helices connected by a flexible loop. Our results suggest that flexibility within the putative helix is important for FDH function and could be a point for regulation of the enzyme.     

Knocking down 10-formyltetrahydrofolate dehydrogenase increased oxidative stress and impeded zebrafish embryogenesis by obstructing morphogenetic movement

Background

Folate is an essential nutrient for cell survival and embryogenesis. 10-Formyltetrahydrofolate dehydrogenase (FDH) is the most abundant folate enzyme in folate-mediated one-carbon metabolism. 10-Formyltetrahydrofolate dehydrogenase converts 10-formyltetrahydrofolate to tetrahydrofolate and CO₂, the only pathway responsible for formate oxidation in methanol intoxication. 10-Formyltetrahydrofolate dehydrogenase has been considered a potential chemotherapeutic target because it was down-regulated in cancer cells. However, the normal physiological significance of 10-Formyltetrahydrofolate dehydrogenase is not completely understood, hampering the development of therapeutic drug/regimen targeting 10-Formyltetrahydrofolate dehydrogenase.

Methods

10-Formyltetrahydrofolate dehydrogenase expression in zebrafish embryos was knocked-down using morpholino oligonucleotides. The morphological and biochemical characteristics of fdh morphants were examined using specific dye staining and whole-mount in-situ hybridization. Embryonic folate contents were determined by HPLC.

Results

The expression of 10-formyltetrahydrofolate dehydrogenase was consistent in whole embryos during early embryogenesis and became tissue-specific in later stages. Knocking-down fdh impeded morphogenetic movement and caused incorrect cardiac positioning, defective hematopoiesis, notochordmalformation and ultimate death of morphants. Obstructed F-actin polymerization and delayed epiboly were observed in fdh morphants. These abnormalities were reversed either by adding tetrahydrofolate or antioxidant or by co-injecting the mRNA encoding 10-formyltetrahydrofolate dehydrogenase N-terminal domain, supporting the anti-oxidative activity of 10-formyltetrahydrofolate dehydrogenase and the in vivo function of tetrahydrofolate conservation for 10-formyltetrahydrofolate dehydrogenase N-terminal domain.

Conclusions

10-Formyltetrahydrofolate dehydrogenase functioned in conserving the unstable tetrahydrofolate and contributing to the intracellular anti-oxidative capacity of embryos, which was crucial in promoting proper cell migration during embryogenesis.

General significance

These newly reported tetrahydrofolate conserving and anti-oxidative activities of 10-formyltetrahydrofolate dehydrogenase shall be important for unraveling 10-formyltetrahydrofolate dehydrogenase biological significance and the drug development targeting 10-formyltetrahydrofolate dehydrogenase.     

Phylogeny and evolution of aldehyde dehydrogenase-homologous folate enzymes

Folate coenzymes function as one-carbon group carriers in intracellular metabolic pathways. Folate-dependent reactions are compartmentalized within the cell and are catalyzed by two distinct groups of enzymes, cytosolic and mitochondrial. Some folate enzymes are present in both compartments and are likely the products of gene duplications. A well-characterized cytosolic folate enzyme, FDH (10-formyltetrahydro-folate dehydrogenase, ALDH1L1), contains a domain with significant sequence similarity to aldehyde dehydrogenases. This domain enables FDH to catalyze the NADP(+)-dependent conversion of short-chain aldehydes to corresponding acids in vitro. The aldehyde dehydrogenase-like reaction is the final step in the overall FDH mechanism, by which a tetrahydrofolate-bound formyl group is oxidized to CO(2) in an NADP(+)-dependent fashion. We have recently cloned and characterized another folate enzyme containing an ALDH domain, a mitochondrial FDH. Here the biological roles of the two enzymes, a comparison of the respective genes, and some potential evolutionary implications are discussed. The phylogenic analysis suggests that the vertebrate ALDH1L2 gene arose from a duplication event of the ALDH1L1 gene prior to the emergence of osseous fish >500 millions years ago.     

10-formyltetrahydrofolate dehydrogenase, one of the major folate enzymes, is down-regulated in tumor tissues and possesses suppressor effects on cancer cells.

Our studies showed that an abundant folate enzyme, 10-formyltetrahydrofolatedehydrogenase (FDH), is strongly down-regulated in several types of cancer on both the mRNA and the protein level. Transient expression of FDH in several human prostate cancer cell lines, a hepatocarcinoma cell line, HepG2, and a lung cancer cell line, A549, suppressed proliferation and resulted in cytotoxicity. In contrast, overexpression of a catalytically inactive FDH mutant did not inhibit proliferation, which suggests that the suppressor effect of FDH is a result of its enzymatic function. Because the FDH substrate, 10-formyltetrahydrofolate, is required for de novo purine biosynthesis, we hypothesized that the inhibitory effects of FDH occur through the depletion of intracellular 10-formyltetrahydrofolate followed by the loss of de novo purine biosynthesis. The ultimate impact is diminished DNA/RNA biosynthesis. Indeed, supplementation of FDH-overexpressing cells with 5-formyltetrahydrofolate or hypoxanthine reversed the FDH growth-inhibitory effects. Hence, down-regulation of FDH in tumors is proposed to be one of the cellular mechanisms that enhance proliferation.     

Aspartate 142 is involved in both hydrolase and dehydrogenase catalytic centers of 10-formyltetrahydrofolate dehydrogenase

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 (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. N(t)-FDH shares some sequence identity with several 10-formyltetrahydrofolate-utilizing enzymes. All these enzymes have a strictly conserved aspartate, which is Asp(142) in the case of N(t)-FDH. Replacement of the aspartate with alanine, asparagine, glutamate, or glutamine in N(t)-FDH resulted in complete loss of hydrolase activity. All the mutants, however, were able to bind folate, although with lower affinity than wild-type N(t)-FDH. Six other aspartate residues located near the conserved Asp(142) 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 Asp(142) 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.     

Classification and enzyme kinetics of formate dehydrogenases for biomanufacturing via CO2 utilization

The reversible interconversion of formate (HCOO^?) and carbon dioxide (CO₂) is catalyzed by formate dehydrogenase (FDH, EC 1.17.1.9). This enzyme can be used as a first step in the utilization of CO₂ as carbon substrate for production of high-in-demand chemicals. However, comparison and categorization of the very diverse group of FDH enzymes has received only limited attention. With specific emphasis on FDH catalyzed CO₂ reduction to HCOO^?, we present a novel classification scheme for FDHs based on protein sequence alignment and gene organization analysis. We show that prokaryotic FDHs can be neatly divided into six meaningful sub-types. These sub-types are discussed in the context of overall structural composition, phylogeny of the gene segment organization, metabolic role, and catalytic properties of the enzymes. Based on the available literature, the influence of electron donor choice on the efficacy of FDH catalyzed CO₂ reduction is quantified and compared. This analysis shows that methyl viologen and hydrogen are several times more potent than NADH as electron donors. Hence, the new FDH classification scheme and the electron donor analysis provide an improved base for developing FDH-facilitated CO₂ reduction as a viable step in the utilization of CO₂ as carbon source for green production of chemicals.     

Modular organization of FDH: Exploring the basis of hydrolase catalysis

An abundant enzyme of liver cytosol, 10-formyltetrahydrofolate dehydrogenase (FDH), is an interesting example of a multidomain protein. It consists of two functionally unrelated domains, an aldehyde dehydrogenase-homologous domain and a folate-binding hydrolase domain, which are connected by an approximately 100-residue linker. The amino-terminal hydrolase domain of FDH (Nt-FDH) is a homolog of formyl transferase enzymes that utilize 10-formyl-THF as a formyl donor. Interestingly, the concerted action of all three domains of FDH produces a new catalytic activity, NADP+-dependent oxidation of 10-formyltetrahydrofolate (10-formyl-THF) to THF and CO2. The present studies had two objectives: First, to explore the modular organization of FDH through the production of hybrid enzymes by domain replacement with methionyl-tRNA formyltransferase (FMT), an enzyme homologous to the hydrolase domain of FDH. The second was to explore the molecular basis for the distinct catalytic mechanisms of Nt-FDH and related 10-formyl-THF utilizing enzymes. Our studies revealed that FMT cannot substitute for the hydrolase domain of FDH in order to catalyze the dehydrogenase reaction. It is apparently due to inability of FMT to catalyze the hydrolysis of 10-formyl-THF in the absence of the cosubstrate of the transferase reaction despite the high similarity of the catalytic centers of the two enzymes. Our results further imply that Ile in place of Asn in the FDH hydrolase catalytic center is an important determinant for hydrolase catalysis as opposed to transferase catalysis.     

On the role of conserved histidine 106 in 10-formyltetrahydrofolate dehydrogenase catalysis: connection between hydrolase and dehydrogenase mechanisms

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 (N(t)-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, His(106), in FDH function. Site-directed mutagenesis experiments showed that replacement of the histidine with alanine, asparagine, aspartate, glutamate, glutamine, or arginine in N(t)-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 N(t)-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 N(t)-FDH. Expressed full-length FDH with the substitution of lysine for the His(106) completely lost both the hydrolase and dehydrogenase activities. Thus, our study showed that His(106), 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.     

FDH: an aldehyde dehydrogenase fusion enzyme in folate metabolism

FDH (10-formyltetrahydrofolate dehydrogenase, Aldh1L1, EC 1.5.1.6) converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate and CO(2) in a NADP(+)-dependent reaction. It is a tetramer of four identical 902 amino acid residue subunits. The protein subunit is a product of a natural fusion of three unrelated genes and consists of three distinct domains. The N-terminal domain of FDH (residues 1-310) carries the folate binding site and shares sequence homology and structural topology with other enzymes utilizing 10-formyl-THF as a substrate. In vitro it functions as 10-formyl-THF hydrolase, and evidence indicate that this activity is a part of the overall FDH mechanism. The C-terminal domain of FDH (residues 400-902) originated from an aldehyde dehydrogenase-related gene and is capable of oxidation of short-chain aldehydes to corresponding acids. Similar to classes 1 and 2 aldehyde dehydrogenases, this domain exists as a tetramer and defines the oligomeric structure of the full-length enzyme. The two catalytic domains are connected by an intermediate linker (residues 311-399), which is a structural and functional homolog of carrier proteins possessing a 4'-phosphopantetheine prosthetic group. In the FDH mechanism, the intermediate linker domain transfers a formyl, covalently attached to the sulfhydryl group of the phosphopantetheine arm, from the N-terminal domain to the C-terminal domain. The overall FDH mechanism is a coupling of two sequential reactions, a hydrolase and a formyl dehydrogenase, bridged by a substrate transfer step. In this mechanism, one domain provides the folate binding site and a hydrolase catalytic center to remove the formyl group from the folate substrate, another provides a transfer vehicle between catalytic centers and the third one contributes the dehydrogenase machinery further oxidizing formyl to CO(2).     

The crystal structure of the hydrolase domain of 10-formyltetrahydrofolate dehydrogenase: mechanism of hydrolysis and its interplay with the dehydrogenase domain

10-Formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyltetrahydrofolate, a precursor for nucleotide biosynthesis, to tetrahydrofolate. The protein comprises two functional domains: a hydrolase domain that removes a formyl group from 10-formyltetrahydrofolate and a NADP(+)-dependent dehydrogenase domain that reduces the formyl to carbon dioxide. As a first step toward deciphering the catalytic mechanism of the enzyme, we have determined the crystal structure of the hydrolase domain of FDH from rat, solved to 2.3-A resolution. The structure comprises two domains. As expected, domain 1 shares the same Rossmann fold as the related enzymes, methionyl-tRNA-formyltransferase and glycinamide ribonucleotide formyltransferase, but, unexpectedly, the structural similarity between the amino-terminal domain of 10-formyltetrahydrofolate dehydrogenase and methionyl-tRNA-formyltransferase extends to the C terminus of both proteins. The active site contains a molecule of beta-mercaptoethanol that is positioned between His-106 and Asp-142 and that appears to mimic the formate product. We propose a catalytic mechanism for the hydrolase reaction in which Asp-142 polarizes the catalytic water molecule and His-106 orients the carbonyl group of formyl. The structure also provides clues as to how, in the native enzyme, the hydrolase domain transfers its product to the dehydrogenase domain.     

10-formyltetrahydrofolate dehydrogenase requires a 4'-phosphopantetheine prosthetic group for catalysis

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 CO(2), 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 CO(2) 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 protein. A 4'-phosphopantetheine swinging arm bound through a phosphoester bond to Ser(354) 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 protein and provides the previously lacking component of the FDH catalytic mechanism.     

Distribution of Folate Derivatives and Enzymes for Synthesis of 10-Formyltetrahydrofolate in Cytosolic and Mitochondrial Fractions of Pea Leaves

Leaf extracts of 14-d-old pea (Pisum sativum L. cv Homesteader) seedlings were examined for folate derivatives and for 10-formyltetrahydrofolate synthetase (SYN), 5,10-methenyltetrahydrofolate cyclohydrolase (CYC), and 5,10-methylenetetrahydrofolate dehydrogenase (DHY) activities. Microbiological and enzyme assays showed that leaf folates SYN, CYC, and DHY were predominantly cytosolic. Extracts of Percoll gradient-purified mitochondria contained less than 1% of total leaf folate and less that 1% of each enzyme activity. Fractionation of whole-leaf homogenates resulted in the copurification of DHY and CYC (subunit 38 kD) and the isolation of a SYN protein (subunit 66 kD). Polyclonal antibodies were raised against purified cytosolic DHY-CYC (DHY-CYC-Ab) and cytosolic SYN (SYN-Ab), respectively. Immunoblots showed that DHY-CYC-Ab cross-reacted with a mitochondrial protein band (38 kD). Two mitochondrial protein bands (subunit Mr = 40,000 and 44,000) cross-reacted with SYN-Ab. Immunoaffinity chromatography (DHY-CYC-Ab as the immobile ligand) indicated that the bulk of mitochondrial SYN activity was not associated with mitochondrial DHY or CYC. When 9-d-old etiolated pea seedlings were exposed to light for up to 3 d, the specific enzyme activities of DHY-CYC in whole-leaf extracts rose 2-fold and more DHY-CYC-Ab cross-reacting protein was detected. In contrast, the specific activity of SYN fell from 5 to 1 [mu]mol min-1 mg-1 protein and less SYN-Ab cross-reacting protein was detected. The data suggest that in pea leaves, the bulk of one-carbon-substituted tetrahydrofolates and enzymes for the generation of 10-formyltetrahydrofolate are extra-mitochondrial.     

C1-Tetrahydrofolate synthase from rabbit liver. Structural and kinetic properties of the enzyme and its two domains

C1-Tetrahydrofolate synthase is a multifunctional enzyme which catalyzes three reactions in 1-carbon metabolism: 10-formyltetrahydrofolate synthetase; 5,10-methenyltetrahydrofolate cyclohydrolase; 5,10-methylenetetrahydrofolate dehydrogenase. A rapid 1-day purification procedure has been developed which gives 40 mg of pure enzyme from 10 rabbit livers. The 10-formyltetrahydrofolate synthetase activity of this trifunctional enzyme has a specific activity that is 4-fold higher than the enzyme previously purified from rabbit liver. Conditions have been developed for the rapid isolation of a tryptic fragment of the enzyme which contains the methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities. This fragment is a monomer exhibiting a subunit and native molecular weight of 36,000 in most buffers. However, in phosphate buffers the native molecular weight suggests that the fragment is a dimer. Conditions are also given whereby chymotryptic digestion allows the simultaneous isolation from the native enzyme of a large fragment containing the 10-formyltetrahydrofolate synthetase activity and a smaller fragment containing the dehydrogenase and cyclohydrolase activities. The large fragment is a dimer with a subunit molecular weight of 66,000. The small fragment retains all of the dehydrogenase and cyclohydrolase activities of the native enzyme. The large fragment is unstable but retains most of the 10-formyltetrahydrofolate synthetase activity. Km values of substrates for the two fragments are the same as the values for the native enzyme. The 10-formyltetrahydrofolate synthetase activity of the native enzyme requires ammonium or potassium ions for expression of full catalytic activity. The effect of these two ions on the catalytic activity of the large chymotryptic fragment is the same as with the native enzyme. We have shown by differential scanning calorimetry that the native enzyme contains two protein domains which show thermal transitions at 47 and 60 degrees C. Evidence is presented that the two domains are related to the two protein fragments generated by proteolysis of the native enzyme. The larger of the two domains contains the active site for the 10-formyltetrahydrofolate synthetase activity while the smaller domain contains the active site which catalyzes the dehydrogenase and cyclohydrolase reactions. Replacement of sodium ion buffers with either ammonium or potassium ions results in an increase in stability of the large domain of the native enzyme. This change in stability is not accompanied by a change in the quaternary structure of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Efficient CO2-Reducing Activity of NAD-Dependent Formate Dehydrogenase from Thiobacillus sp. KNK65MA for Formate Production from CO2 Gas

NAD-dependent formate dehydrogenase (FDH) from Candida boidinii (CbFDH) has been widely used in various CO₂-reduction systems but its practical applications are often impeded due to low CO₂-reducing activity. In this study, we demonstrated superior CO₂-reducing properties of FDH from Thiobacillus sp. KNK65MA (TsFDH) for production of formate from CO₂ gas. To discover more efficient CO₂-reducing FDHs than a reference enzyme, i.e. CbFDH, five FDHs were selected with biochemical properties and then, their CO₂-reducing activities were evaluated. All FDHs including CbFDH showed better CO₂-reducing activities at acidic pHs than at neutral pHs and four FDHs were more active than CbFDH in the CO₂ reduction reaction. In particular, the FDH from Thiobacillus sp. KNK65MA (TsFDH) exhibited the highest CO₂-reducing activity and had a dramatic preference for the reduction reaction, i.e., a 84.2-fold higher ratio of CO₂ reduction to formate oxidation in catalytic efficiency (k _cat/K _B) compared to CbFDH. Formate was produced from CO₂ gas using TsFDH and CbFDH, and TsFDH showed a 5.8-fold higher formate production rate than CbFDH. A sequence and structural comparison showed that FDHs with relatively high CO₂-reducing activities had elongated N- and C-terminal loops. The experimental results demonstrate that TsFDH can be an alternative to CbFDH as a biocatalyst in CO₂ reduction systems.     

Regulation of folate-mediated one-carbon metabolism by 10-formyltetrahydrofolate dehydrogenase

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes the NADP(+)-dependent conversion of 10-formyltetrahydrofolate to CO(2) and tetrahydrofolate (THF) and is an abundant high affinity folate-binding protein. Although several activities have been ascribed to FDH, its metabolic role in folate-mediated one-carbon metabolism is not well understood. FDH has been proposed to: 1) inhibit purine biosynthesis by depleting 10-formyl-THF pools, 2) maintain cellular folate concentrations by sequestering THF, 3) deplete the supply of folate-activated one-carbon units, and 4) stimulate the generation of THF-activated one-carbon unit synthesis by channeling folate cofactors to other folate-dependent enzymes. The metabolic functions of FDH were investigated in neuroblastoma, which do not contain detectable levels of FDH. Both low and high FDH expression reduced total cellular folate concentrations by 60%, elevated rates of folate catabolism, and depleted cellular 5-methyl-THF and S-adenosylmethionine levels. Low FDH expression increased the formyl-THF/THF ratio nearly 10-fold, whereas THF accounted for nearly 50% of total folate in neuroblastoma with high FDH expression. FDH expression did not affect the enrichment of exogenous formate into methionine, serine, or purines and did not suppress de novo purine nucleotide biosynthesis. We conclude that low FDH expression facilitates the incorporation of one-carbon units into the one-carbon pool, whereas high levels of FDH expression deplete the folate-activated one-carbon pool by catalyzing the conversion of 10-formyl-THF to THF. Furthermore, FDH does not increase cellular folate concentrations by sequestering THF in neuroblastoma nor does it inhibit or regulate de novo purine biosynthesis. FDH expression does deplete cellular 5-methyl-THF and S-adenosylmethionine levels indicating that FDH impairs the folate-dependent homocysteine remethylation cycle.     

Isolation and characterization of cDNA clones for rat liver 10-formyltetrahydrofolate dehydrogenase.

We have isolated and characterized cDNA clones encoding rat liver cytosol 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6). An open reading frame of 2706 base pairs encodes for 902 amino acids of Mr 99,015. The deduced amino acid sequence contains exact matches to the NH2-terminal sequence (28 residues) and the sequences of five peptides derived from cyanogen bromide cleavage of the purified protein. The amino acid sequence of 10-formyltetrahydrofolate dehydrogenase has three putative domains. The NH2-terminal sequence (residues 1-203) is 24-30% identical to phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) from Bacillus subtilis (30%), Escherichia coli (24%), Drosophila melanogaster (24%), and human hepatoma HepG2 (27%). Residues 204-416 show no extensive homology to any known protein sequence. Sequence 417-900 is 46% (mean) identical to the sequences of a series of aldehyde dehydrogenase (NADP+) (EC 1.2.1.3). Intact 10-formyltetrahydrofolate dehydrogenase exhibits NADP-dependent aldehyde dehydrogenase activity. The sequence identity to phosphoribosylglycinamide formyltransferase is discussed, and a binding region for 10-formyltetrahydrofolate is proposed.     

Crystal structures of the carboxyl terminal domain of rat 10-formyltetrahydrofolate dehydrogenase: implications for the catalytic mechanism of aldehyde dehydrogenases

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes an NADP+-dependent dehydrogenase reaction resulting in conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. This reaction is a result of the concerted action of two catalytic domains of FDH, the amino-terminal hydrolase domain and the carboxyl-terminal aldehyde dehydrogenase domain. In addition to participation in the overall FDH mechanism, the C-terminal domain is capable of NADP+-dependent oxidation of short chain aldehydes to their corresponding acids. We have determined the crystal structure of the C-terminal domain of FDH and its complexes with oxidized and reduced forms of NADP. Compared to other members of the ALDH family, FDH demonstrates a new mode of binding of the 2'-phosphate group of NADP via a water-mediated contact with Gln600 that may contribute to the specificity of the enzyme for NADP over NAD. The structures also suggest how Glu673 can act as a general base in both acylation and deacylation steps of the reaction. In the apo structure, the general base Glu673 is positioned optimally for proton abstraction from the sulfur atom of Cys707. Upon binding of NADP+, the side chain of Glu673 is displaced from the active site by the nicotinamide ring and contacts a chain of highly ordered water molecules that may represent a pathway for translocation of the abstracted proton from Glu673 to the solvent. When reduced, the nicotinamide ring of NADP is displaced from the active site, restoring the contact between Cys707 and Glu673 and allowing the latter to activate the hydrolytic water molecule in deacylation.     

Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is found in both the cytoplasm and the mitochondria. The gene encoding yeast mitochondrial C1-tetrahydrofolate synthase was isolated using synthetic oligonucleotide probes based on the amino-terminal sequence of the purified protein. Hybridization analysis shows that the gene (designated MIS1) has a single copy in the yeast genome. The predicted amino acid sequence of mitochondrial C1-tetrahydrofolate synthase shares 71% identity with yeast C1-tetrahydrofolate synthase and shares 39% identity with clostridial 10-formyltetrahydrofolate synthetase. Chromosomal deletions of the mitochondrial C1-tetrahydrofolate synthase gene were generated using the cloned MIS1 gene. Mutant strains which lack a functional MIS1 gene are viable and can grow in medium containing a nonfermentable carbon source. In fact, deletion of the MIS1 locus has no detectable effect on cell growth.